Immunogenic conjugates of Gram-negative bacterial autoinducer molecules and antibodies raised against the same

ABSTRACT

The present invention relates to an immunogenic conjugate comprising a carrier molecule coupled to an autoinducer of a Gram negative bacteria. The immunogenic conjugate, when combined with a pharmaceutically acceptable carrier, forms a suitable vaccine for mammals to prevent infection by the Grain negative bacteria. The immunogenic conjugate is also used to raise and subsequently isolate antibodies or binding portions thereof which are capable of recognizing and binding to the autoinducer. The antibodies or binding portions thereof are utilized in a method of treating infections, a method of inhibiting autoinducer activity, and in diagnostic assays which detect the presence of autoinducers or autoinducer antagonists in fluid or tissue samples.

[0001] This application is a divisional of U.S. patent application Ser.No. 09/293,687, filed Apr. 16, 1999, which claims priority to U.S.Provisional Patent Application Serial No. 60/082,025, filed Apr. 16,1998, which is incorporated herein in its entirety.

GOVERNMENT SUPPORT

[0002] This research was supported by grant number AI133713-04 from theNational Institute of Health.

1. FIELD OF THE INVENTION

[0003] The present invention relates to an immunogenic conjugatecomprising an autoinducer molecule of a Gram negative bacteria or asynthetic analogue thereof linked to a carrier molecule. The presentinvention also relates to antibodies and binding portions thereofcapable of binding the immunogenic conjugate, and vaccines for thetreatment or prevention of infection by autoinducer producing bacteria.

2. BACKGROUND OF THE INVENTION

[0004] Some pathogenic Gram negative bacteria produce chemical moietiesknown as bacterial autoinducers (BAIs). BAIs are produced by Gramnegative bacteria as a mechanism for communicating with other bacteriawhen they have grown to a high cell density. This mechanism is known asquorum sensing.

[0005] BAIs assist in the transcriptional control of genes involved in awide range of metabolic activities. When the bacterial populationreaches a critical threshold, the concentration of BAIs also reach aconcentration sufficient to enable the BAIs to bind a group oftranscription factors, known as R-proteins. Binding of the BAIs to theR-proteins triggers binding of the newly formed BAI/R-protein complex toDNA, which then induces transcription of a group of genes. In the gramnegative pathogenic bacteria, a subgroup of the activated genes arepathogenic determinants.

[0006] BAIs are small, non-imnmunogenic, lipid-soluble molecules whichare capable of diffusing out of the bacteria, and into the environmentwhere they enter host cells. BAIs share structural characteristics, inparticular, they have a homoserine lactone ring with an N-acyl sidechain. Variability between different BAIs resides primarily in thestructure of the acyl side chain.

[0007] It has been proposed that in addition to regulating transcriptionin bacteria, the BAIs also regulate transcription in cells of aninfected mammalian host. A list of presently known bacterialautoinducers (BAIs) and the Gram negative bacteria which produce themare identified in Table 1 below: TABLE 1 Gram negative bacteria:Bacterial autoinducer (BAI): Aeromonas hydrophila AHAI Agrobacteriumtumefaciens N-(3-oxo)-octanoyl-L-homoserine lactone (OOHL) Burkholderiacepacia N-octanoylhomoserine lactone Chromobacterium violaceumN-hexanoyl-L-homoserine lactone (HHL) Enterobacter agglomeransN-(3-oxo)-hexanoyl-L-homoserine lactone (OHHL) Erwinia stewarti OHHLErwinia carotovora OHHL Escherichia coli Structure not yet determinedNitrosomas europea OHHL Photobacterium fischeri OHHL, OOHL; OHLPseudomonas aeruginosa N-(3-oxododecanoyl)-L-homoserine lactone (PAI-1);N-(butanoyl)-L-homoserine lactone (PAI-2 Pseudomonas aureofaciensStructure not yet determined Rhizobium leguminosarumN-(3-hydroxy)-tetradecanoyl-L- homoserine lactone (HtDeHL) Serratialiquefaciens PAI-2 Vibrio fischeri OHHL Vibric harveyiN-(3-hydroxy)-butanoyl-L-homoserine lactone (HBHL) Yersiniaenterocolitica OHHL, HHL

[0008] The Gram negative bacterium Pseudomonas aeruginosa is anopportunistic human pathogen that causes infections in immunocompromisedhosts. PAI-1 has been shown to inhibit the proliferation of lymphocytesin vivo and downregulates expression of tumor necrosis factor andinterleukin-12 (Telford et al., 1998, Infect Immun. 66(1):36-42).Pseudomonas aeruginosa frequently colonizes the lungs of individualswith cystic fibrosis (Hoiby, N., 1974, Acta Pathologica Microbiolo.Scand. Sect. B. 82:551-558; Reynolds et al., 1975, Ann. Intern. Med.82:819-832). This bacterium produces a number of extracellular virulencefactors including exotoxin A, which is encoded by the toxA gene(Iglewski, B. H. and Kabat, D., 1975, Proc. Natl. Acad. Sci. USA.72:2284-2288; Iglewski et al., 1978, Proc. Natl. Acad. Sci. USA.75:3211-3215); an elastolytic protease encoded by the lasA gene; anelastolytic protease encoded by the lasB gene; and an alkaline proteaseencoded by the aprA gene (Morihara, K. and Homma, J. Y., 1985, BacterialEnzymes and Virulence, ed. Holder, I. A. (CRC Press, Boca Raton, Fla.)pp. 41-79; Bever, R. A. and Iglewski, B. H., 1988, J. Bacteriol.170:4309-4313; Kessler, E. and Saffrin, M., 1988, J. Bacteriol.170:5241-5247).

[0009]Pseudomonas aeruginosa utilizes a partially redundant quorumsensing mechanism which includes two autoinducers,N-(3-oxododecanoyl)-L-homoserine lactone (PAI-1) andN-(butanoyl)-L-homoserine lactone (PAI-2) (see Table 1). Theseautoinducers control expression of a number of virulence factors,including the elastolytic proteases lasA and lasB, autoinducer synthase,alkaline protease, exotoxin A and rhamnolipid synthase. (Garnbello, etal., 1993, Infection & Immunity 61:1180-84; Latifi, et al., 1996, Mol.Microbiol. 21:1137-46; Passador, et al., 1993, Science 260:1127-30;Pesci, et al., 1997, J. Bact. 179:3127-32; Seed, et. al., 1995, J. Bact.177:654-59; and Toder, et al., 1994, Infection & Immunity 62:1320-27.)It is the production of these virulence factors which enable Pseudomonasaeruginosa to invade and induce disease in humans.

[0010] Current treatments for Gram negative bacterial infectionstypically target surface antigens of the bacteria to make antibodies.Development of vaccines and diagnostic antibodies to autoinducers arehindered by the fact that autoinducers are not only non-immunogenic, butare also freely diffusible through the lipid bilayer and are notcovalently attached to the bacteria. Several studies have demonstratedthat a non-immunogenic bacterial capsular polysaccharide may beconjugated to an immunogenic compound to generate antibodies to thecapsular polysaccharide (Anderson, U.S. Pat. No. 4,673,574 (conjugationof a fragment of a bacterial capsular polymer to a diphtheria or tetanustoxin or toxoid); Wessels et al., 1990, J. Clin. Invest. 86:1428-1433(conjugation of a polysaccharide of type III group B Streptococcus totetanus toxoid); and Schneerson et al., 1980, J. Exp. Med. 152:361-376(conjugation of H. influenzae type b capsular polysaccharide to tetanustoxoid and other carriers.)) However, in contrast to autoinducers whichare lipid diffusible, these anti-polysaccharide treatments are designedfor production of antibodies specific to surface antigens covalentlyattached to the bacteria, resulting in lysis of the bacteria.

[0011] While synthetic autoinducer analogs limit bacterial growth invitro, this approach fails to harness the capabilities of an activeimmune response that is a potentially long-lasting and effectivetherapeutic or prophylactic treatment (Pearson et al., U.S. Pat. No.5,591,872). Furthermore, although autoinducer molecules themselves canbe used in diagnostic bioassays, including bioluminescence, antibioticproduction, or bacterial growth, these diagnostic assays fail to providea prophylactic or therapeutic benefit to individuals exposed toautoinducer-producing Gram negative bacteria (Bycroft et al., U.S. Pat.No. 5,593,827).

3. SUMMARY OF THE INVENTION

[0012] The present invention relates to immunogenic conjugatescomprising a carrier molecule covalently conjugated or otherwise boundto an autoinducer of a Gram negative bacteria of a compound of Formula(1):

[0013] where X is O, S, N—(C₁-C₆) alkyl, NR², N-phenyl; Y is C₁-C₆straight or branched alkyl, C₁-C₆ straight or branched alkenyl, C₁-C₆straight or branched alkynyl; Z is C═O, C═S, CHOH, C═N—NR¹, C═N—OH,C₁-C₈ straight or branched alkyl, C₁-C₈ straight or branched alkenyl,C₁-C₈ straight or branched alkynyl; L is C₁-C₁₈ straight or branchedalkyl, C₁-C₁₈ straight or branched alkenyl, C₁-C₁₈ straight branchedalkynyl, or —CO₂H, —CO₂R¹, —CHO, —C≡N, —N═C═O, —N═C═S, OH, OR¹,—CH═CH—CH₂Br, —CH═CH—CH₂Cl, —SAc or SH, where R¹ is C₁-C₆ straight orbranched alkyl, m is 0 or 1; z is 0 or 1; R² is H, C₁-C₆ straight orbranched alkyl, C₁-C₆ straight or branched alkenyl or C₁-C₆ straight orbranched alkynyl, or CO₂H; and Q is CH or N; and n is 0-3 with theproviso that when n is 0, X is N—C₁-C₆ alkyl) or N-phenyl. In a specificembodiment, the carrier molecule comprises a lysine-containing protein,preferably, including but not limited to bovine serum albumin, chickenegg ovalbumin, keyhole limpet hemocyanin, tetanus toxoid, diphtheriatoxoid, and thyroglobulin.

[0014] In specific embodiments, the autoinducer is produced by a Gramnegative bacteria comprising Aeromonas hydrophila, Agrobacteriumtumefaciens, Burkholderia cepacia, Chromobacterium violaceum,Enterobacter agglomerans, Erwinia stewarti, Erwinia carotovora,Escherichia coli, Nitrosomas europea, Photobacterium fischeri,Pseudomonas aeruginosa, Pseudomonas aureofaciens, Rhizobiumleguminosarum, Serratia liquefaciens, or Vibrio harveyi.

[0015] In specific embodiments, the autoinducer comprisesN-(3-oxododecanoyl)-L-homoserine lactone, N-(butanoyl)-L-homoserinelactone, N-hexanoyl-homoserine lactone, N-(3-oxohexanoyl)-homoserinelactone, N-β(hydroxybutyryl)-homoserine lactone,N-(3-oxooctanoyl)-L-homoserine lactone, orN-(3R-hydroxy-cis-tetradecanoyl)-L-homoserine lactone, preferably,N-(3-oxododecanoyl)-L-homoserine lactone (PAI-1) orN-(butanoyl)-L-homoserine lactone (PAI-2).

[0016] In a specific embodiment, the carrier molecule of the immunogenicconjugate has at least one amine group, the autoinducer has an N-acylhomoserine lactone structure, and the conjugate is the reductiveamination product of the carrier molecule and the autoinducer.

[0017] The invention also relates to isolated antibodies or fragmentsthereof which specifically bind an autoinducer produced by a Gramnegative bacteria. In an embodiment, the autoinducer is a compound ofFormula (I) (described above). In another embodiment, the autoinducercomprises N-(3-oxododecanoyl)-L-homoserine lactone,N-(butanoyl)-L-homoserine lactone, N-hexanoyl-homoserine lactone,N-(3-oxohexanoyl)-homoserine lactone, N-β(hydroxybutyryl)-homoserinelactone, N-(3-oxooctanoyl)-L-homoserine lactone, orN-(3R-hydroxy-cis-tetradecanoyl)-L-homoserine lactone. In a specificembodiment, the autoinducer is N-(3-oxododecanoyl)-L-homoserine lactoneor N-(butanoyl)-L-homoserine lactone.

[0018] The invention also relates to isolated antibodies or fragmentsthereof which specifically bind an autoinducer produced by a Gramnegative bacteria in which the autoinducer is covalently conjugated orotherwise bound to a carrier molecule. The carrier molecule includes butis not limited to bovine serum albumin, chicken egg ovalbumin, keyholelimpet hemocyanin, tetanus toxoid, diphtheria toxoid, and thyroglobulin.

[0019] In specific embodiments, the autoinducer which is specificallybound by the antibodies or fragments thereof of the invention isproduced by a Gram negative bacteria comprising Aeromonas hydrophila,Agrobacterium tumefaciens, Burkholderia cepacia, Chromobacteriumviolaceum, Enterobacter agglomerans, Erwinia stewarti, Erwiniacarotovora, Escherichia coli, Nitrosomas europea, Photobacteriumfischeri, Pseudomonas aeruginosa, Pseudomonas aureofaciens, Rhizobiumleguminosarum, Serratia liquefaciens, or Vibrio harveyi.

[0020] The invention also relates to methods for detecting a Gramnegative bacteria autoinducer in a sample comprising adding to thesample an antibody in which the antibody specifically binds theautoinducer of a Gram negative bacteria of a compound of Formula (I)(described above). In an embodiment, the autoinducer is produced by aGram negative bacteria including but not limited to Aeromonashydrophila, Agrobacterium tuinetaciens, Burkholderia cepacia,Chromobacterium violaceum, Enterobacter agglomerans, Erwinia stewarti,Erwinia carotovora, Escherichia coli, Nitrosomas europea, Photobacteriumfischeri, Pseudomonas aeruginosa, Pseudomonas aureofaciens, Rhizobiumleguminosarum, Serratia liquefaciens, or Vibrio harveyi.

[0021] The invention also relates to methods of treating or preventingan infectious disease in a subject comprising administering an amount ofan immunogenic conjugate in which the immunogenic conjugate comprises acarrier molecule covalently conjugated or otherwise bound to anautoinducer of a Gram negative bacteria of a compound of Formula (I)(described above); preferably, the subject is a human.

[0022] The invention also relates to methods of treating or preventingan infectious disease in a subject comprising administering an amount ofan antibody or fragment thereof which specifically binds an autoinducerof a Gram negative bacteria of a compound of Formula (I) (describedabove); preferably, the subject is a human.

[0023] The invention also relates to diagnostic kits and pharmaceuticalcompositions comprising the immunogenic conjugates or antibodies orfragments thereof of the invention.

[0024] The present invention also relates to methods of inhibitingautoinducer activity. The methods comprise contacting an effectiveamount of the antibody or binding portion thereof with an autoinducerunder conditions effective to bind the autoinducer in which the amountis effective to treat or prevent infection by Gram negative bacteria.

4. BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1. Comparison of the role of autoinducers PAI-1 and PAI-2 inthe virulence of Pseudomonas aeruginosa. Neonatal mice were infectedwith Pseudomonas aeruginosa strains PAO1(wild type), PAO-JP2 (bearingthe lasI/rhII double deletion), PAO-JP1 (bearing lasI single deletion),and PAO-JP2/pJPP42 (bearing the lasI/rhII double deletion with plasmidexpressing lasI/rhII). The mice were sacrificed and examined forpneumonia (black bars), bacteremia (light gray bars), and mortality(dark gray bars).

[0026]FIG. 2. The effect of anti-PAI-1 murine polyclonal antibodies onan E. coli transcriptional bioassay. A 1:10 dilution of mouse serum waspreincubated with 100 pM PAI-1 prior to addition to the bioassay. “Mal1” is compound D conjugate immune serum, “PAI-1” is PAI-1 conjugateimmune serum, “Pre” is serum from mice prior to any immunization and“control” is PAI-1 without addition of serum. Addition of immune serumexhibited a 70% inhibition in the production of β-galactosidase.

[0027]FIG. 3. The effect of anti-PAI-1 monoclonal antibodies in aPseudomonas aeruginosa transcriptional bioassay. When an anti-PAI-1monoclonal antibody (618.4) was added to 40 nM PAI-1 prior to additionto the bioassay an 80% inhibition in β-galactosidase production wasexhibited.

5. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention provides for the production of immunogenicconjugates of autoinducers and immunogenic conjugates for use asvaccines or for production of antibodies useful for immunotherapy ordiagnostic assays.

[0029] Solely for ease of explanation, the description of the inventionis divided into the following sections: (A) isolation of homoserinelactones; (B) conjugation of autoinducers to immunogenic carriers; (C)generation of antibodies to BAI immunogenic conjugates; (D) therapeuticuses of immunogenic conjugates or antibodies thereto; (E) diagnosticassays.

[0030] 5.1 Isolation and Synthesis of Homoserine Lactones

[0031] Autoinducers of Gram negative bacteria can be isolated andpurified by various methods known in the art. Purification of homoserinelactones through chemical methods can be used to isolate naturallyproduced autoinducers. The autoinducer N-(β-ketocaproyl) homoserinelactone has been purified from a culture supernatant of Erwiniacarotovora (Bycroft et al., U.S. Pat. No. 5,593,827, hereby incorporatedby reference). Following bacterial culture centrifugation, theautoinducer is extracted from the supernatant using ethyl acetate, theresulting sample is mixed with water and the ethyl acetate removed.Next, the sample is passed through a column containing a hydrophobicresin which is eluted with a methanol in water solution to remove theautoinducer. The methanol in water solution, now containing theautoinducer, is concentrated by rotary evaporation. Thereafter, theautoinducer is purified using HPLC and additional rotary evaporation.

[0032] A similar process has been developed for purifying the naturallyoccurring autoinducer of Pseudomonas aeruginosa,N-(3-oxododecanoyl)-L-homoserine lactone (PAI-1) (Pearson et al., U.S.Pat. No. 5,591,872, which is hereby incorporated by reference). First,cells and culture fluid are separated by centrifugation and the culturefluid subsequently passed through a 0.2 μm pore-size filter. Thefiltered material is repeatedly extracted using alternatingethanol/ethyl acetate steps. The sample is subsequently dissolved inmethanol and purified using HPLC with reverse phase column. Additionalextraction and purification by HPLC yield the isolated autoinducerPAI-1.

[0033] Eberhard et al., 1981, Biochem. 20:2444-49, which is herebyincorporated by reference, describes a process for chemicallysynthesizing N-(3-oxohexanoyl)-homoserine lactone. U.S. Pat. No.5,591,872 to Pearson et al., establishes that the process of Eberhard etal. may also be used to synthesize N-(3-oxododecanoyl)-L-homoserinelactone (PAI-1), by using a different starting material (i.e., ethyl3-oxododecanoate rather than ethyl 3-oxohexanoate). Therefore, it may beappreciated by one of ordinary skill in the art that the procedure ofEberhard et al. may be used to synthesize a variety of autoinducermolecules and their derivatives by utilizing different startingmaterials.

[0034] Compounds of the Formula (I) where L is CO₂H or CO₂-alkyl, m is0, Y is CH₂, n is 1, Q is CH, and X is O may be prepared according toScheme I. Reaction of L-homoserine lactone hydrochloride (B) withmalonic acid mono tert-butylester (A) in methylene chloride at roomtemperature in the presence of an organic base, such as triethylamine,and a coupling agent, such as benzotriazol-1-yloxytris (dimethylamino)phosphonium hexafluorophosphate yielded the corresponding amide (C).Amide (C) was deprotected under acidic conditions using trifluoroaceticacid (TFA) in methylene chloride at room temperature to yield thecarboxylic acid (D). Homoserine derivatives of the formula (D) can beconjugated to amine or alcohol functionalities on the appropriatecarrier protein by reaction with the carboxylic acid moiety understandard conditions to one skilled in the art to form an amide or esterconjugate, respectively, with the carrier protein.

[0035] Compounds of the Formula (I) where Y and L are alkyl, Q is CH, nis 1 and m is 0 were prepared in a single step as outlined in Scheme 2.Thus, reaction of the sodium or lithium salts of the fatty acids 1-3with optically pure L-homoserine lactone hydrochloride (Sigma ChemicalCo., St. Louis, Mo.) in the presence of the commercially available watersoluble coupling agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (Aldrich Chemical Company, Milwaukee, Wis.), provided thewaxy amides 5-7 in good yield. In each case, the crude products wereconveniently purified by recrystallization from ethyl acetate/hexane.

[0036] The natural form of Pseudomonas autoinducer (PAI) 13 wassynthesized in a novel six step sequence as outlined in Scheme 3.Standard acylation of Meldrums acid with decanoyl chloride in thepresence of pyridine provided the acyl Meidrums species 8. Reaction of 8with methanol under reflux conditions then provided the beta-ketoester 9in good yield. The C3 carbonyl group of 9 was then protected as itsketal under standard conditions and hydrolysis of the methyl esterfunctionality under basic conditions then afforded the carboxylate 10.Coupling of 10 and L-homoserine lactone hydrochloride under aqueousconditions in the presence of water soluble carbodiimide 4 then providedthe amide 11 in good yield. Final deprotection of 11 was accomplishedwith dilute acid affording the target keto-amide 13 (PAI). An analogoussequence was carried out in order to prepare the keto-amide 14 bearing athiolactone moiety. In this sequence, optically pure L-homocysteinethiolactone hydrochloride (available from Sigma) was substituted forL-homoserine lactone hydrochloride in the carbodiimide mediated couplingstep. The resulting dioxolane-amide 12 was then deprotected with diluteacid to provide the keto-amide 14.

[0037] Compounds of the Formula (I) where Z is CHOH, Y is CH₂, L isalkyl, n is 1, Q is CH and X is 0 were prepared by reduction of the C3keto group of 13 as outlined in Scheme 4. Thus, low temperaturereduction of 13 with sodium borohydride in methanol provided adiastereomeric mixture of alcohols 15.

[0038] Compounds of the Formula (I) where X is 0, Q is N, m is 0, z is0, and R² is straight or branched alkyl were prepared by directacylation of 16 with decanoyl chloride (Scheme 4). Deprotonation ofcommercially available optically pure 16 with BuLi followed by quenchingthe resulting anion with decanoyl chloride, provided the acylatedoxazolidinone 17 in good yield.

[0039] Compounds of the Formula (I) where X is 0, Y is CH₂, Z is C═O, Qis CH, n is 1, and L is C₁-C₁₈ straight or branched alkenyl may beprepared by the method of Scheme 5. Careful oxidation of commerciallyavailable trans-4-decanal with sodium hypochlorite in the presence ofsulfamic acid (as chlorine scavenger), provided crude acid 18 which wasnot purified, but converted directly to the acid chloride 19 with oxalylchloride and DMF(cat.). Reaction of 19 with Meldrums acid in thepresence of pyridine provided the acyl Meldrums species 20. Heatingcrude 20 in methanol then afforded the beta-ketoester 21 which was thenpurified by flash chromatography. Protection of the ketone carbonylgroup of 21 as its ketal followed by hydrolysis of the ester withlithium hydroxide gave the carboxylate salt 23. Coupling of 23 andL-Homoserine lactone hydrochloride in the presence of the carbodiimide 4in aqueous medium, provided the amide 24. Finally, deprotection of 24under acidic conditions gave the unsaturated keto-amide 25 in 36% yieldover 3 steps.

[0040] Compounds of the Formula (I) where X is NH, Q is CH, Y is CH₂, Zis C═O, L is alkyl, n is 1, m is 1, and Z is C═O were prepared accordingto Scheme 6. The keto-lactam 28 was prepared as outlined in Scheme 6.Hydrolysis of 10 with lithium hydroxide followed by carbodiimidemediated coupling with aminolactam hydrochloride 26 provided theprotected lactam 27. The aminolactam hydrochloride 26 was prepared fromcommercially available (S)-(+)-2,4-Diaminobutyric acid hydrochloride(Aldrich Chemical Co.) according to the literature procedure. D. W.Adamson, J. Chem. Soc. 1943:39-41. Hydrolysis of 27 with dilute acidthen gave the target keto-lactam 28 in good overall yield.

[0041] Compounds of the Formula (I) where Y is C₁-C₆ alkenyl, m is 0,and Z is CO₂H can be made by the reaction of homoserine lactone withmaleic anhydride in methylene chloride at room temperature to yieldcompound 29. (Scheme 7). Linshitz, Y., et al., J. Amer. Chem. Soc., 77,1265-6 (1955).

[0042] The carboxylic acid moiety can then be coupled to a carrierprotein containing a free amino or hydroxy group under standardconditions to yield the corresponding immunogenic conjugate.

[0043] Autoinducer molecules suitable for use in forming immunogenicconjugates of the present invention are the autoinducers of Gramnegative bacteria of a compound of Formula (I) and derivatives thereof.In particular, the autoinducers used to form the immunogenic conjugateof the present invention are the autoinducers of the following Gramnegative bacteria: Aeromonas hydrophiia, Agrobacterium tumefaciens,Burkholderia cepacia, Chromobacterium violaceum, Enterobacteragglomerans, Erwinia stewarti, Erwinia carotovora, Escherichia coli,Nitrosomas europea, Photobacterium fischeri, Pseudomonas aeruginosa,Pseudomonas aureofaciens, Rhizobium leguminosarum, Serratialiqueffaciens, and Vibrio harveyi.

[0044] Preferred autoinducer molecules areN-(3-oxododecanoyl)-L-homoserine lactone, N-(butyryl)-L-homoserinelactone, N-butanoyl-L-homoserine lactone, N-hexanoyl-homoserine lactone,N-(3-oxohexanoyl)-homoserine lactone, N-β-(hydroxybutyryl)-homoserinelactone, N-(3-oxooctanoyl)-L-homoserine lactone,N-(3R-hydroxy-cis-tetradecanoyl)-L-homoserine lactone, other N-acylhomoserine lactones, their derivatives and analogs.

[0045] The structures of various exemplary autoinducer molecules areshown below:

[0046] 5.2 Bacterial Autoinducer Conjugation to a Carrier

[0047] Once the desired autoinducer is isolated, the immunogenicconjugates of the present invention are formed by coupling the carriermolecule to the autoinducer. Typically, these reactions are conducted byreacting available hydroxy or amino groups in a protein, with a compoundof the formula (I) as shown below:

[0048] (I)

[0049] where X is O, S, N—(C₁-C₆) alkyl, NR², N-phenyl; Y is C₁-C₆straight or branched alkyl, C₁-C₆ straight or branched alkenyl, C₁-C₆straight or branched alkynyl; Z is C═O, C═S, CHOH, C═N—NR¹, C═N—OH,C₁-C₈ straight or branched alkyl, C₁-C₈ straight or branched alkenyl,C₁-C₈ straight or branched alkynyl; L is C₁-C₁₈ straight or branchedalkyl, C₁-C₁₈ straight or branched alkenyl, C₁-C₁₈ straight branchedalkynyl, or CO₂H, —CO₂R¹, CHO, —C≡N, —N═C═O, —N═C═S, OH, OR¹,—CH═CH═CH₂Br, —CH═CH—CH₂Cl, —SAc or SH, where R¹ is C₁-C₆ straight orbranched alkyl, m is 0 or 1; z is 0 or 1; R² is H, C₁-C₆ straight orbranched alkyl, C₁-C₆ straight or branched alkenyl or C₁-C₆ straight orbranched alkynyl, or CO₂H; and Q is CH, or N; and n is 0-3 with theproviso that when n is 0, X is N—(C₁-C₆ alkyl) or N-phenyl.

[0050] The reactions are performed under acidic or basic conditions inthe presence of a suitable coupling agent. For example, an autoinducerof Gram negative bacteria may contain a reactive ketone moiety in acompound of Formula (I). The reaction of the reactive ketone moiety of acompound of Formula (I) with a carrier molecule such as a proteincontaining a free amino group will produce a Schiff base which can thenbe reduced with sodium cyanoborohydride to produce the correspondingamine (reductive amination). Sodium cyanoborohydride is the preferredagent for the reduction of Schiff bases in the presence of other ketonemoieties present in the autoinducer compound of Formula (I).

[0051] Other reducing agents can be used to reductively aminate ketonemoieties instead of sodium cyanoborohydride. Their suitability willdepend upon the functionalities present in the autoinducer compound ofFormula (I). The alternative reducing agents include hydrogen and acatalyst of either sodium triacetoxyborohydride (Carson et al., 1990,Tetrahedron Letters, 31, 5595), sodium borohydride (Schellenberg, 1963,J. Org. Chem., 28, 3259) alcoholic potassium hydroxide (Watanabe etal.,, 1974, Tetrahedron Letters, 1879) or BH₃-Pyridine (Pelter et al.,1984, J. Chem. Suc. Perkin Trans. 1:717). The reductive aminationprocess is carried out using the method set forth by Schwartz and Gray,“Proteins Containing Reductively Aminated Disaccharides: Synthesis andChemical Characterization,” Arch. Biochem. Biophys. 181:542-549 (1977),which is hereby incorporated by reference.

[0052] Alternatively, a carrier molecule such as a protein containing afree amino group can be coupled to carbon-carbon multiple bonds presentin a compound of Formula (I) under basic conditions to form an amineconjugate or imine conjugate in the case where addition occurs to analkyne. The free amino group of a protein could also add to anisocyanate or isothiocyanate moiety of a compound of Formula (I) toyield a urea or thiourea conjugate, respectively. Vishnyakora, et al.,Russ. Chem. Rev. 54, 249-261 (1985). Additionally, amine conjugatecontaining a compound of Formula (I) can be made by reaction of anallylic halide moiety with a protein containing a free amino group underbasic conditions.

[0053] For example, the Pseudomonas aeruginosa autoinducer PAI-1 may beconjugated to a carrier molecule such as a lysine containing protein(i.e., bovine serum albumin). PAI-1 and the protein carrier molecule aremixed at a 1:10 molar ratio in phosphate buffer (pH 7.0). PAI-1, via theketo group, interacts with the terminal amino groups of lysines on thesurface of the protein carrier molecule to form a Schiff base. Themixture is incubated until conjugation is substantially complete. Afterconjugation, the intermediate Schiff base formed between PAI-1 and theprotein carrier molecule is reduced with sodium cyanoborohydride.Finally, the mixture is dialyzed for removal of unbound PAI-1 andresidual cyanoborohydride.

[0054] Alternatively, a BAI may be prepared by coupling a carboxylicacid moiety of L or R² of a compound of Formula (I) with an amine of aprotein to form a peptide conjugate. As coupling reagents, one can useany standard peptide coupling activation method for carboxylic acidmoieties such as those exemplified in schemes 1-6 described earlier, orother coupling agents or methods (e.g., photocoupling) known to thoseskilled in the art.

[0055] As another alternative, the BAI may be non-covalently bound tothe carrier molecule by being absorbed to the carrier molecule usingtechniques commonly known in the art.

[0056] Suitable carrier molecules are those which are safe foradministration to mammals and immunologically effective as carriers.Safety would include the absence of primary toxicity and minimal risk ofallergic complications. Preferred carrier molecules are bovine serumalbumin, chicken egg ovalbumin, keyhole limpet hemocyanin, tetanustoxoid, diphtheria toxoid, thyroglobulin, and other lysine containingproteins. Each of the preferred carrier molecules fulfill thesecriteria, because they are non-toxic and the incidence of allergicreaction is well known.

[0057] The formation of immunogenic conjugates comprising an autoinducerof a Gram negative bacteria and a carrier molecule enables a wide arrayof therapeutic and/or prophylactic agents and diagnostic procedures for,respectively, treating or preventing infection by Gram negative bacteriaand detecting the presence of autoinducer molecules produced thereby.

[0058] 5.3 Generation of Antibodies to BAI Immunogenic Conjugates

[0059] Another aspect of the present invention relates to isolatedantibodies or binding portions thereof which specifically bind anautoinducer of the present invention. The present invention alsoencompasses pharmaceutical compositions comprising the antibodies orbinding portions thereof and a pharmaceutically acceptable carrier.

[0060] An autoinducer immunogenic conjugate or autoinducer or derivativethereof, may be used as an immunogen to generate antibodies whichrecognize the said immunogen. The antibodies include but are not limitedto polyclonal antibodies, monoclonal antibodies (mAbs), humanized orchimeric antibodies, single chain antibodies, Fab fragments, F(ab′)₂fragments, fragments produced by a Fab expression library,anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments ofany of the above.

[0061] The antibodies or binding portions thereof, or compositionscontaining the same, are useful in treating mammals, preferably humans,exposed to or otherwise infected with a autoinducer producing Gramnegative bacteria. Methods of treatment using the compositions of thepresent invention include but are not limited to passive immunotherapy,idiotype vaccination, etc. Methods of treatment encompassed by thepresent invention comprise administration of a therapeutically effectiveamount of the antibody or binding portion thereof in which the antibodyor binding portion thereof is capable of binding an autoinducer of aGram negative bacteria.

[0062] Various procedures known in the art may be used for theproduction of polyclonal antibodies to an autoinducer immunogenicconjugate. For the production of antibodies, various host animals can beimmunized by injection with an immunogenic conjugate, or a syntheticversion, or derivative (e.g., fragment) thereof. Such host animalsinclude but are not limited to rabbits, mice, rats, etc. Adjuvants maybe used to increase the immunological response, depending on the hostspecies, including but not limited to Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanins, dinitrophenol, andpotentially useful human adjuvants such as BCG (bacille Calmette-Guerin)and Corynebacterium parvum. Various procedures for raising polyclonalantibodies are described in E. Harlow, et al., editors, Antibodies: ALaboratory Manual (1988), which is hereby incorporated by reference. Foran illustrative example, see section 6.2, infra.

[0063] For preparation of monoclonal antibodies directed toward anautoinducer or analog thereof, any technique which provides for theproduction of antibody molecules by continuous cell lines in culture maybe used. For example, the hybridoma technique of Kohler and Milstein(1975, Nature 256:495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc., pp. 77-96). Human antibodies may be used and can be obtained byusing human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030) or by transforming human B cells with EBV virus in vitro(Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, pp. 77-96). In addition, monoclonal antibodies can be produced ingerm-free animals utilizing recent technology (PCT/US90/02545).

[0064] In addition, techniques developed for the production of “chimericantibodies” (Morrison, et al., 1984, Proc. Natl. Acad. Sci.,81,6851-6855; Neuberger, et al., 1984, Nature 312,604-608; Takeda, etal., 1985, Nature, 314,452-454, incorporated herein by reference intheir entirety) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used, forexample, the genes from a mouse antibody molecule specific for anautoinducer can be spliced together with genes from a human antibodymolecule of appropriate biological activity. A chimeric antibody is amolecule in which different portions are derived from different animalspecies, such as those having a variable region derived from a murinemAb and a human immunoglobulin constant region. (See, e.g., Cabilly etal., U.S. Pat. No. 4,816,567; and Boss et al., U.S. Pat. No. 4,816,397,which are incorporated herein by reference in their entirety.)

[0065] In addition, techniques have been developed for the production ofhumanized antibodies. (See, e.g., Queen, U.S. Pat. No. 5,585,089 andWinter, U.S. Pat. No. 5,225,539, which are incorporated herein byreference in their entirety.) An immunoglobulin light or heavy chainvariable region consists of a “framework” region interrupted by threehypervariable regions, referred to as complementarity determiningregions (CDRs). The extent of the framework region and CDRs have beenprecisely defined (see, “Sequences of Proteins of ImmunologicalInterest”, Kabat, E. et al., U.S. Department of Health and HumanServices (1983), incorporated hereinby reference in their entirety).Briefly, humanized antibodies are antibody molecules from non-humanspecies having one or more CDRs from the non-human species and aframework region from a human immunoglobulin molecule.

[0066] Alternatively, techniques described for the production of singlechain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science242,423-426; Huston, et al., 1988, Proc. Natl. Acad. Sci. USA85,5879-5883; and Ward, et al., 1989, Nature 334,544-546, incorporatedherein by reference in their entirety) can be adapted to produce singlechain antibodies against an immunogenic conjugate of the presentinvention. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide.

[0067] In another embodiment, the methods of the present inventionencompass use of antibody fragments comprising the idiotype of the wholeantibody. Such fragments include but are not limited to: the F(ab′)₂fragments, which can be produced by pepsin digestion of the antibodymolecule and the Fab fragments, which can be generated by reducing thedisulfide bridges of the F(ab′)₂ fragments (J. Goding, MonoclonalAntibodies: Principles and Practice, pp. 98-118 (N.Y. Academic Press1983), which is incorporated herein by reference). Alternatively, theFab fragments can be generated by treating the antibody molecule withpapain and a reducing agent. Alternatively, Fab expression libraries maybe constructed (Huse, et al., 1989, Science, 246:1275-1281, incorporatedherein by reference in its entirety) to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity.

[0068] In an embodiment of the invention, molecules comprising thebinding portion of antibodies which specifically bind an autoinducer orthe epitope of an autoinducer may be used in the methods of theinvention. Such molecules include peptides, derivatives and analogsthereof, and peptide mimetics.

[0069] The autoinducer specific antibodies may be isolated by standardtechniques known in the art such as immunoaffinity chromatography,centrifugation, precipitation, etc. Screening for the desired antibodycan be accomplished by techniques known in the art including but notlimited to, competitive and non-competitive assay systems usingtechniques such as radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoradiometric assays, gel diffusionprecipitin reactions, immonodiffusion assays, in situ immunoassays(using colloidal gold, enzyme or radioisotope labels, for example),western blots, precipitation reactions, agglutination assays (e.g., gelagglutination assays, hemagglutination assays), complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled.

[0070] The foregoing antibodies can be used for treating or preventingdiseases caused by the autoinducer producing Gram negative bacteria orfor detecting and measuring the activity of the autoinducer of theinvention, e.g., for imaging these autoinducers, measuring levelsthereof in appropriate physiological samples, in diagnostic assays, etc.as discussed in section 5.5 infra.

[0071] The antibodies generated by the vaccine formulations of thepresent invention can also be used in the production of antiidiotypicantibody. The antiidiotypic antibody can then in turn be used forimmunization, in order to produce a subpopulation of antibodies thatbind the initial antigen of the pathogenic microorganism (Jerne, 1974,Ann. Immunol. (Paris) 125c:373; Jerne, et al., 1982, EMBO J. 1:234).

[0072] 5.4 Therapeutic Uses of Immunogenic Conjugates or AntibodiesThereto

[0073] Autoinducer immunogenic conjugates and antibodies whichspecifically bind an autoinducer by a Gram negative bacteria can be usedfor treating or preventing an infectious disease caused by a Gramnegative bacteria.

[0074] 5.4.1 Administration and Formulation

[0075] Immunogenic conjugates comprising an autoinducer of a Gramnegative bacteria of a compound of Formula (I) coupled to a carriermolecule can be used as vaccines for immunization against saidautoinducer producing Gram negative bacteria. The vaccines, comprisingthe immunogenic conjugate in a pharmaceutically acceptable carrier, areuseful in a method of immunizing mammals, preferably humans, fortreatment or prevention of infections by the said autoinducer producingGram negative bacteria.

[0076] The antibodies generated against the autoinducer immunogenicconjugate of the present invention by immunization with the autoinducerimmunogenic conjugate can be used in passive immunotherapy andgeneration of antiidiotypic antibodies for treating or preventinginfectious disease caused by a Gram negative bacteria.

[0077] In a specific embodiment, an immunogenic conjugate comprising thePseudomonas aeruginosa autoinducer PAI-1 (see Table 1) is administeredas a vaccine to Pseudomonas aeruginosa. In another embodiment, animmunogenic conjugate comprising the Pseudomonas aeruginosa autoinducerPAI-2 (see Table 1) is administered as a vaccine. In another embodiment,an immunogenic conjugate comprising PAI-1 is administered before,during, or after administration of an immunogenic conjugate comprisingPAI-2. In another embodiment, an autoinducer immunogenic conjugatevaccine is administered before, during or after administration of aneffective amount of an antibody or binding portion thereof which hasbeen raised against an immunogenic conjugate of the type describedabove.

[0078] In am embodiment, an antibody or binding portion thereof whichspecifically binds an immunogenic conjugate comprising PAI-1 isadministered. In another embodiment, an antibody or binding portionthereof which specifically binds PAI-2 is administered. In anotherembodiment the antibody which specifically binds PAI-1 is administeredbefore, during or after administration of an antibody which specificallybinds PAI-2.

[0079] Methods of administration of the compositions of the invention(i.e., BAI immunogenic conjugate vaccine or antibodies specific to theBAI immunogenic conjugates) include but are not limited to oral,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal routes, and scarification (scratching through the top layersof skin, e.g., using a bifurcated needle).

[0080] The patient to which the composition is administered ispreferably a mammal, including but not limited to cows, horses, sheep,pigs, fowl (e.g. chickens), goats, cats, dogs, hamsters, mice and rats.In a preferred embodiment the subject is a human.

[0081] The formulations of the invention comprise an effectiveimmunizing amount of one or more autoinducer immunogenic conjugate orantibody thereto and a pharmaceutically acceptable carrier or excipient.Subunit vaccines comprise an effective immunizing amount of one or moreAntigens and a pharmaceutically acceptable carrier or excipient.Pharmaceutically acceptable carriers are well known in the art andinclude but are not limited to saline, buffered saline, dextrose, water,glycerol, sterile isotonic aqueous buffer, and combinations thereof. Oneexample of such an acceptable carrier is a physiologically balancedculture medium containing one or more stabilizing agents such asstabilized, hydrolyzed proteins, lactose, etc. The carrier is preferablysterile. The formulation should suit the mode of administration.

[0082] The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. The compositioncan be a liquid solution, suspension, emulsion, tablet, pill, capsule,sustained release formulation, or powder. Oral formulation can includestandard carriers such as pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, etc.

[0083] Generally, the ingredients are supplied either separately ormixed together in unit dosage form, for example, as a dry lyophilizedpowder or water free concentrate in a hermetically sealed container suchas an ampule or sachette indicating the quantity of active agent. Wherethe composition is administered by injection, an ampule of sterilediluent can be provided so that the ingredients may be mixed prior toadministration.

[0084] Peptides, derivatives and analogs thereof, and peptide mimeticsthat specifically bind an autoinducer can be produced by various methodsknown in the art, including, but not limited to solid-phase synthesis orby solution (Nakanishi et al., 1993, Gene 137:51-56; Merrifield, 1963,J. Am. Chem. Soc. 15:2149-2154; Neurath, H. et al., Eds., The Proteins,Vol II, 3d Ed., p. 105-237, Academic Press, New York, N.Y. (1976),incorporated herein in their entirety by reference).

[0085] Suitable carriers include lubricants and inert fillers such aslactose, sucrose, or cornstarch. In another embodiment, these compoundsare tableted with conventional tablet bases such as lactose, sucrose, orcornstarch in combination with binders like acacia, gum gragacanth,cornstarch, or gelatin; disintegrating agents such as cornstarch, potatostarch, or alginic acid; a lubricant like stearic acid or magnesiumstearate; and sweetening agents such as sucrose, lactose, or saccharine;and flavoring agents such as peppermint oil, oil of wintergreen, orartificial flavorings.

[0086] The autoinducer immunogenic conjugates or the autoinducerantibodies or binding portions thereof of the present invention may alsobe administered in injectable dosages by solution or suspension of thesematerials in a physiologically acceptable diluent with a pharmaceuticalcarrier. Such carriers include sterile liquids such as water and oils,with or without the addition of a surfactant and other pharmaceuticallyacceptable adjuvants. Illustrative oils are those of petroleum, animal,vegetable, or synthetic origin, for example, peanut oil, soybean oil, ormineral oil. In general, water, saline, aqueous dextrose and relatedsugar solution, and glycols such as polypropylene glycol or polyethyleneglycol, are preferred liquid carriers, particularly for injectablesolutions. To maintain sterility and prevent action of microorganisms,antibacterial and antifungal agents, such as parabens, chlorobutanol,phenol, ascorbic acid, thimerosal, and the like may be added to thecarrier.

[0087] For use as aerosols, the immunogenic conjugates of the presentinvention, or antibodies or binding portions thereof according to thepresent invention, are alveolar lavage, lymph nodes, bone marrow, orother biopsied materials.

[0088] Toxicity and therapeutic efficacy of such molecules can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population).

[0089] The vaccines of the invention may be multivalent or univalent.Multivalent vaccines are made from immuno-conjugation of multipleautoinducers with a carrier molecule.

[0090] In an embodiment, the autoinducer immunogenic conjugate vaccineformulation comprises an effective immunizing amount of the autoinducerimmunogenic conjugate, preferably in combination with animmunostimulant; and a pharmaceutically acceptable carrier. As used inthe present context, “immunostimulant” is intended to encompass anycompound or composition which has the ability to enhance the activity ofthe immune system, whether it be a specific potentiating effect incombination with a specific antigen, or simply an independent effectupon the activity of one or more elements of the immune response.Immunostimulant compounds include but are not limited to mineral gels,e.g., aluminum hydroxide; surface active substances such aslysolecithin, pluronic polyols; polyanions; peptides; oil emulsions;alum, and MDP. Methods of utilizing these materials are known in theart, and it is well within the ability of the skilled artisan todetermine an optimum amount of stimulant for a given autoinducervaccine. More than one immunostimulant may be used in a givenformulation. The immunogen may also be incorporated into liposomes, orconjugated to polysaccharides and/or other polymers for use in a vaccineformulation.

[0091] The compositions may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredient. The pack may for example comprisemetal or plastic foil, such as a blister pack. The pack or dispenserdevice may be accompanied by instructions for administration preferablyfor administration to a human. Associated with such container(s) can bea notice in the form prescribed by a governmental. agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

[0092] 5.4.2 Effective Dose

[0093] The compounds described herein can be administered to a patientat therapeutically effective doses to treat certain diseases caused byGram negative bacteria that cause infectious diseases. A therapeuticallyeffective dose refers to that amount of a compound sufficient to resultin a healthful benefit in the treated subject.

[0094] The precise amount of immunogenic conjugate or antibody whichspecifically binds a BAI to be employed in the formulation will dependon the route of administration and the nature of the patient (e.g., age,size, stage/level of disease), and should be decided according to thejudgment of the practitioner and each patient's circumstances accordingto standard clinical techniques. An effective immunizing amount is thatamount sufficient to treat or prevent an infectious disease caused by aGram negative bacteria that produces an autoinducer in a subject.Effective doses may also be extrapolated from dose-response curvesderived from animal model test systems and can vary from 0.001 mg/kg to100 mg/kg.

[0095] Toxicity and therapeutic efficacy of compounds can be determinedby standard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LDs₅₁/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

[0096] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

[0097] Immunopotency of a composition can be determined by monitoringthe immune response of test animals following immunization with thecomposition by use of any immunoassay known in the art. Generation of ahumoral (antibody) response and/or cell-mediated immunity, may be takenas an indication of an immune response. Test animals may include mice,hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., andeventually human subjects.

[0098] The immune response of the test subjects can be analyzed byvarious approaches such as: the reactivity of the resultant immune serumto the immunogenic conjugate, as assayed by known techniques, e.g.,enzyme linked immunosorbent assay (ELISA), immunoblots,immunoprecipitations, etc.; or, by protection of immunized hosts frominfection by the pathogen and/or attenuation of symptoms due toinfection by the pathogen in immunized hosts as determined by any methodknown in the art, for assaying the levels of an infectious diseaseagent, e.g., the bacterial levels (for example, by culturing of a samplefrom the patient), etc. The levels of the infectious disease agent mayalso be determined by measuring the levels of the antigen against whichthe immunoglobulin was directed. A decrease in the levels of theinfectious disease agent or an amelioration of the symptoms of theinfectious disease indicates that the composition is effective.

[0099] The therapeutics of the invention can be tested in vitro, andthen in vivo, for the desired therapeutic or prophylactic activity,prior to use in humans. For example, in vitro assays that can be used todetermine whether administration of a specific therapeutic is indicatedinclude in vitro cell culture assays in which appropriate cells from acell line or cells cultured from a patient having a particular diseaseor disorder are exposed to or otherwise administered a therapeutic, andthe effect of the therapeutic on the cells is observed.

[0100] Alternatively, the therapeutic may be assayed by contacting thetherapeutic to cells (either cultured from a patient or from a culturedcell line) that are susceptible to infection by the infectious diseaseagent but that are not infected with the infectious disease agent,exposing the cells to the infectious disease agent, and then determiningwhether the infection rate of cells contacted with the therapeutic waslower than the infection rate of cells not contacted with thetherapeutic. Infection of cells with an infectious disease agent may beassayed by any method known in the art.

[0101] In addition, the therapeutic can be assessed by measuring thelevel of the molecule against which the antibody is directed in theanimal model or human subject at suitable time intervals before, during,or after therapy. Any change or absence of change in the amount of themolecule can be identified and correlated with the effect of thetreatment on the subject. The level of the molecule can be determined byany method known in the art as described supra.

[0102] After vaccination of an animal to a Gram negative autoinducerusing the methods and compositions of the present invention, any bindingassay known in the art can be used to assess the binding between theresulting antibody and the particular molecule. These assays may also beperformed to select antibodies that exhibit a higher affinity orspecificity for the particular antigen.

[0103] 5.5 Detection and Diagnostic Methods

[0104] Autoinducer antibodies or binding portions of the presentinvention are useful for detecting in a sample the presence of anautoinducer of a Gram negative bacteria. This detection method comprisesthe steps of providing an isolated antibody or binding portion thereofraised against an autoinducer of a Gram negative bacteria, adding to theisolated antibody or binding portion thereof a sample suspected ofcontaining a quantity of the autoinducer, and detecting the presence ofa complex comprising the isolated antibody or binding portion thereofbound to the autoinducer.

[0105] The antibodies or binding portions thereof of the presentinvention are also useful for detecting in a sample the presence of anautoinducer antagonist. This detection method comprises the steps ofproviding an isolated antibody or binding portion thereof raised againstan autoinducer antagonist, adding to the isolated antibody or bindingportion thereof a sample suspected of containing a quantity of theautoinducer antagonist, and detecting the presence of a complexcomprising the isolated antibody or binding portion thereof bound to theautoinducer antagonist.

[0106] Immunoglobulins, particularly antibodies, (and functionallyactive fragments thereof) that bind a specific molecule that is a memberof a binding pair may be used as diagnostics and prognostics, asdescribed herein. In various embodiments, the present invention providesthe measurement of a member of the binding pair, and the uses of suchmeasurements in clinical applications. The immunoglobulins in thepresent invention may be used, for example, in the detection of anantigen in a biological sample whereby patients may be tested foraberrant levels of the molecule to which the immunoglobulin binds,and/or for the presence of abnormal forms of such molecules. By“aberrant levels” is meant increased or decreased relative to thatpresent, or a standard level representing that present, in an analogoussample from a portion of the body or from a subject not having thedisease. The antibodies of this invention may also be included as areagent in a kit for use in a diagnostic or prognostic technique.

[0107] In an embodiment of the invention, an antibody of the inventionthat immunospecifically binds to an infectious disease agent may be usedto diagnose, prognose or screen for an infectious disease associatedwith the expression of the antigen of the infectious disease agent.

[0108] In a preferred aspect, the invention provides a method ofdiagnosing or screening for the presence of an infectious disease agent,characterized by the presence of an autoinducer antigen of a Gramnegative bacteria of said infectious disease agent, comprising measuringin a subject the level of immunospecific binding of an antibody to asample derived from the subject, in which said antibodyimmunospecifically binds said antigen in which an increase in the levelof said immunospecific binding, relative to the level of saidimmunospecific binding in an analogous sample from a subject not havingthe infectious disease agent, indicates the presence of said infectiousdisease agent.

[0109] The measurement of a molecule that is bound by an antibody can bevaluable in detecting and/or staging diseases related to the molecule ina subject, in screening of such diseases in a population, indifferential diagnosis of the physiological condition of a subject, andin monitoring the effect of a therapeutic treatment on a subject.

[0110] Examples of suitable assays to detect the presence ofautoinducers or antagonists thereof include but are not limited toELISA, radioimmunoassay, gel-diffusion precipitation reaction assay,immunodiffusion assay, agglutination assay, fluorescent immunoassay,protein A immunoassay, or immunoelectrophoresis assay.

[0111] The following assays are designed to detect molecules to whichthe antibodies inumunospecifically bind. The tissue or cell type to beanalyzed will generally include those which are known, or suspected, toexpress the particular molecule. The protein isolation methods employedherein may, for example, be such as those described in Harlow and Lane(Harlow, E. and Lane, D., 1988, “Antibodies: A Laboratory Manual”, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The isolatedcells can be derived from cell culture or from a patient. The antibodies(or functionally active fragments thereof) useful in the presentinvention may, additionally, be employed histologically, as inimmunofluorescence, immunohistochemistry, or immunoelectron microscopy,for in situ detection of the molecule. In situ detection may beaccomplished by removing a histological specimen from a patient, such asparaffin embedded sections of affected tissues and applying thereto alabeled antibody of the present invention. The antibody (or functionallyactive fragment thereof) is preferably applied by overlaying the labeledantibody onto a biological sample. If the molecule to which the antibodybinds is present in the cytoplasm, it may be desirable to introduce theantibody inside the cell, for example, by making the cell membranepermeable. Through the use of such a procedure, it is possible todetermine not only the presence of the particular molecule, but also itsdistribution in the examined tissue. Using the present invention, thoseof ordinary skill will readily perceive that any of a wide variety ofhistological methods (such as staining procedures) can be modified inorder to achieve such in situ detection of Gram negative bacteriaautoinducers.

[0112] Immunoassays for the particular molecule will typically compriseincubating a sample, such as a biological fluid, a tissue extract,freshly harvested cells, or lysates of cultured cells, in the presenceof a detectably labeled antibody and detecting the bound antibody by anyof a number of techniques well-known in the art.

[0113] The biological sample may be brought in contact with andimmobilized onto a solid phase support or carrier such asnitrocellulose, or other solid support which is capable of immobilizingcells, cell particles or soluble proteins. The support may then bewashed with suitable buffers followed by treatment with the detectablylabeled antibody. The solid phase support may then be washed with thebuffer a second time to remove unbound antibody. The amount of boundlabel on solid support may then be detected by conventional means.

[0114] “Solid phase support or carrier” includes any support capable ofbinding an antigens or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

[0115] The binding activity of a given antibody may be determinedaccording to well known methods. Those skilled in the art will be ableto determine operative and optimal assay conditions for eachdetermination by employing routine experimentation.

[0116] One of the ways in which an antibody can be detectably labeled isby linking the same to an enzyme and use in an enzyme immunoassay (EIA)(Voller, A., “The Enzyme Linked Immunosorbent Assay (ELISA)”, 1978,Diagnostic Horizons 2:1-7, Microbiological Associates QuarterlyPublication, Walkersville, Md.); Voller et al., 1978, J Clin. Pathol.31:507-520; Butler, 1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.),1980, Enzyme Immunoassay, CRC Press, Boca Raton, Fla.,; Ishikawa et al.,(eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo)). The enzyme whichis bound to the antibody will react with an appropriate substrate,preferably a chromogenic substrate, in such a manner as to produce achemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. The detection can be accomplishedby colorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

[0117] Detection may also be accomplished using any of a variety ofother inmmunoassays. For example, by radioactively labeling thesynthetic antibodies or fragments, it is possible to detect the proteinthat the antibody was designed for through the use of a radioimmunoassay(RIA) (see, for example, Weintraub, 1986, Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society). The radioactive isotope can bedetected by such means as the use of a gamma counter or a scintillationcounter or by autoradiography.

[0118] It is also possible to label the antibody with a fluorescentcompound. When the fluorescently labeled antibody is exposed to light ofthe proper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

[0119] The antibody can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA).

[0120] The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during. the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

[0121] Likewise, a bioluminescent compound may be used to label thesynthetic antibody of the present invention. Bioluminescence is a typeof chemiluminescence found in biological systems, in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin.

[0122] An additional aspect of the present invention relates todiagnostic kits for the detection or measurement of autoinducer immuno.Kits for diagnostic use are provided, that comprise in one or morecontainers an anti-autoinducer antibody, and, optionally, a labeledbinding partner to the antibody. Alternatively, the anti-autoinducerantibody can be labeled (with a detectable marker, e.g., achemiluminescent, enzymatic, fluorescent, or radioactive moiety).Accordingly, the present invention provides a diagnostic kit comprising,an anti-autoinducer antibody and a control immunoglobulin. In a specificembodiment, one of the foregoing compounds of the container can bedetectably labeled. A kit can optionally further comprise in a containera predetermined amount of an autoinducer recognized by the saidanti-autoinducer antibody of the kit, for use as a standard or control.

[0123] The Examples set forth below are for illustrative purposes onlyand are not intended to limit, in any way, the scope of the presentinvention.

6 EXAMPLE Synthesis Preparation of Compound C

[0124] To a stirred solution of malonic acid mono tert-butylester A (419mg, 90% purity, 2.35 mmol) and L-homoserine lactone hydrochloride B (324mg, 2.35 mmol) in CH₂Cl₂ (15 mL) at room temperature was addedsequentially Et₃N (660 μL, 4.94 mmol) andBenzotriazol-1-yloxytris(dimethylamino)phosphonium hexfluorophosphate(BOP) (1.14 g, 2.58 mmol). After it was stirred for 3 hours (h), themixture was concentrated, dissolved in EtOAc, washed with 1M HCl andsaturated NaHCO₃, dried over MgSO₄, filtered, concentrated and theresidual was chromatographed (SiO₂, Hexanes-EtOAc 1:1) to give C as agum (571 mg, 2.35 mmol, ca. 100%).

[0125]¹H NMR (300 MHZ, CDCl₃): δ 7.85 (br s, 1H), 4.61 (m, 1H), 4.50 (m,1H), 4.30 (m, 1H), 3.32 (s, 2H), 2.81 (m, 1H), 2.23 (m, 1H), 1.50 (s,9H). IR (cm⁻¹): 1780, 1716, 1683. HRMS calculated for (C₁₁H₁₇NO₅+NH₄+)at 261.1450, found at 261.1453.

Preparation of Compound D

[0126] To a stirred solution of C (122 mg, 500 μmol) in CH₂C₁₂ (5 mL) atroom temperature was added TFA (385 μL, 5 mmol), and the resultingmixture was stirred for 4 h. The mixture was concentrated to give D as agum (94 mg, 500 μmol, ca. 100%).

[0127]¹H NMR (300 MHZ, CD₃CN): δ 9.70 (br s, 1H), 7.46 (br s, 1H), 4.60(m, 1H), 4.40 (m, 1H), 4.25 (m, 1H), 3.35 (s, 2H), 2.55 (m, 1H), 2.25(m, 1H). IR (cm⁻¹): 1773; 1731; 1656. HRMS calculated for (C₇H₉NO₅+NH₄⁺) at 205.0824, found at 205.0829.

Preparation of Compound 5

[0128] To a stirred solution of L-homoserine lactone (Sigma, 1.0 mmol)in 10 mL of water at room temperature was added the lithium or sodiumsalt of the fatty acid (1.0 mmol) followed by1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (Aldrich,1.0 mmol). After 24 h, the resulting milky suspension was extractedseveral times with ethyl acetate. The combined extracts were dried(MgSO₄), filtered and the solvent removed in vacuo. The crude amideswere then further purified by recrystallization from ethylacetate/hexane.

[0129]¹H NMR (CDCl₃, δ): 6.00 (bm, 1H), 4.46-4.69 (m, 2H), 4.25-4.34 (m,1H), 2.94-2.93 (m, 1H), 2.26 (t, J=7 Hz, 2H), 2.10-2.28 (m, 1H),1.63-1.68 (m, 2H), 1.27-1.31 (m, 10H), 09.89 (t, J=7 Hz, 3H). Anal.calcd for C₁₃H₂₃NO₃: C, 64.68; H, 9.62. Found: C, 64.77; H, 9.72. [α]²⁵_(D)+12⁰ (c=2, CHCl₃), mp. 135-136° C.

Preparation of Compound 6

[0130]¹H NMR (acetone-d₆,+): 7.44 (bm, 1H), 4.55-4.65 (m, 1H), 4.21-4.40(m, 2H), 2.49-2.57 (m, 1H), 2.16-2.27 (m, 3H), 1.53-1.60 (m, 2H), 1.26(s, 16H), 0.85 (t, J=7 Hz, 3H). Anal. calcd for C₁₆11₂₉N0₃: C, 67.79; H,10.33. Found: C, 67.87; H, 10.66.

Preparation of Compound 7

[0131]¹H NMR (CDCl₃, δ): 6.00 (bd, 1H), 4.45-4.60 (m, 2H), 4.25-4.34 (m,1H), 2.84-2.93 (m, 1H), 2.26 (t, J=7 Hz, 2H), 2.09-2.28 (m, 1H),1.62-1.68 (m, 2H), 1.26-1.30 (m, 22H), 0.89 (t, J=7 Hz, 3H). Anal. calcdfor C₁₉H₃₅NO₃: C, 70.01; H, 10.86. Found: C, 69.48; H, 11.20. [α]²⁵_(D)+12⁰ (c=2, CHCl₃), mp. 138.139° C.

Preparation of Compound 8

[0132] To a stirred solution of Meldrums acid* (4.00 g; 27.8 mmol) andpyridine (5.62 mL; 69.4 mmol) in dry CH₂Cl₂ (12 mL) under N₂ at 0° C.was added freshly distilled decanoyl chloride (Aldrich, 5.76 mL; 27.8mmol) dropwise. The reaction mixture gradually became deep red in color.After 1 h the mixture was allowed to warm up to room temperature for 1.5h. At this point, the mixture was poured over crushed ice containing 20mL of 2N HCl. The phases were separated and the aqueous layer furtherextracted with CH₂Cl₂. The combined organic extracts were dried (MgSO₄),filtered and concentrated in vacuo to give 3.882 g of crudebeta-ketoester 8.

[0133]¹H NMR (CDCl₃, δ): 3.03 (m, 2H), 1.75 (s, 6H), 1.62-1.70 (m, 2H),1.25-1.35 (m, 12H), 0.89 (t, J=7 Hz, 3H).

[0134] *It is important to recrystallize the Meldrums acid from acetonebefore carrying out this experiment. The commercial material may bequite impure and adversely affect the purity of the final acylatedproduct.

Preparation of Compound 10

[0135] The crude beta-ketoester from above was dissolved in 20 mL of drymethanol. The mixture was warmed to reflux under N₂. After 2.5 h, themixture was allowed to cool to room temperature and the methanol removedin vacuo. The oily residue was chromatographed (Eluent: 7% ethylacetate/hexane) to give 1.889 g (60%) of the beta-ketoester 9 as amixture of keto-enol tautomers. To a stirred solution of the β-ketoester9 (1.889 g; 8.3 mmol) in 20 mL of benzene at room temperature was addedethylene glycol (1.39 mL; 25 mmol) followed by 20 mg of p-TSA. Theresulting mixture was then warmed to reflux (Dean-Stark). After 24.5 h,the mixture was allowed to cool to room temperature and washed with 10%Na₂CO₃ solution. The organic layer was then dried (MgSO₄), filtered andconcentrated in vacuo. Flash chromatography on silica gel (eluent: 10%ethyl acetate/hexane) provided 1.926g (85%) of the corresponding ketal10 as a clear oil.

[0136]¹H NMR (CDCl₃, δ): 3.99 (m, 4H), 3.70 (s, 3H), 2.68 (s, 2H),1.77-1.82 (m, 2H), 1.35-1.41 (m, 2H), 1.27 (s, 14H), 0.89 (t, J=7 Hz,3H). anal. calcd. for C₁₅H₂₈O₄: C, 66.13; H, 10.38. Found: C, 66.30; H,10.18.

Preparation of Compound 11

[0137] To a stirred solution of the ketal of 9 in methanol at roomtemperature was added LiOH solution (3.65 mL of 1.0 mmol/mL stocksolution). The mixture was then warmed to reflux for 10 min. and thenallowed to cool to room temperature. The solvent was then removed invacuo to give crude carboxylate 10. The crude salt was then dissolved in50 mL of water and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (844 mg; 4.40 mmol) and L-homoserine lactone (556 mg; 4.04mmol) were added sequentially. The mixture was allowed to stir for 6 hat room temperature and then the resulting thick white suspensionextracted several times with ethyl acetate. The combined extracts weredried (MgSO₄), filtered and concentrated in vacuo to give a crudeoff-white solid. Recrystallization from ethyl acetate/hexane afforded752 mg (60%) of the amide 11.

[0138]¹H NMR (CDCl₃, δ): 7.01 (d, J=6 Hz, 1H), 4.56-4.61 (m, 1H), 4.47(t, J=9 Hz, 1H), 4.23-4.32 (m, 1H), 3.98-4.11 (m, 4H), 2.76-2.83 (m,1H), 2.65 (s, 2H), 2.05-2.23 (m, 1H), 1.66-1.75 (m, 4H), 1.26-1.42 (m,2H), 1.26 (s, 10H), 0.88 (t, 3=7 Hz, 3H). [α]²⁵ _(D−)16⁰(c=2, EtOH).

Preparation of Compound 13

[0139] To a stirred solution of the ketal 11 (330 mg; 0.97 mmol) in 2.0mL of methanol at room temperature was added 0.1N HCl solution (2.0 mL).The resulting cloudy mixture was warmed to reflux for 1 h and thenallowed to cool to room temperature. The majority of the methanol wasthen removed in vacuo and the remaining solution extracted several timeswith ethyl acetate. The combined extracts were dried (MgSO₄), filteredand concentrated in vacuo. Flash chromatography on silica gel (eluent:70% ethyl acetate/hexane) afforded 122 mg (43%) of PAI 13 as a waxywhite solid mp. 88-90° C.

[0140]¹H NMR (CDCl₃, δ): 7.70 (bm, 1H), 4.58-4.64 (m, 1H), 4.49 (t, J=9Hz, 1H), 4.25-4.35 (m, 1H), 3.48 (s, 2H), 2.75-2.82 (m, 1H), 2.53 (t,J=6 Hz, 2H), 2.19-2.29 (m, 1H), 1.57-1.65 (m, H), 1.27 (s, H), 0.89 (t,J=9 Hz, 3H). Anal. calcd for C₁₆H₂₇O₄N:C, 64.60; H, 9.17. Found: C,64.60; H, 9.44. [α]²⁵ _(D)−19⁰ (c=2, EtOH).

Preparation of Compound 12

[0141] To a stirred solution of the ester 10 (1.00 g; 3.67 mmol) in 5.0mL of methanol at room temperature was added LiOH solution (3.55 mL of1.0 mmol/mL stock solution). The mixture was then warmed to reflux for15 min. and then allowed to cool to room temperature. The solvent wasthen removed in vacuo, the residue redissolved in 40 mL of water andL-homocysteine thiolactone hydrochloride (473 mg; 3.08 mmol) and1-(3-dimethylaminopropyl)=3=ethylcarbodiimide hydrochloride (590 mg;3.08 mmol) were added sequentially. The resulting mixture was allowed tostir for 19.5 h at room temperature and then extracted several timeswith ethyl acetate. The combined extracts were dried (MgSO₄), filteredand concentrated in vacuo. Flash chromatography on silica gel (eluent:85% ethyl acetate/hexane) provided 585 mg. (64%) of thethiolactone-amide 12 as a white solid.

[0142]¹H NMR (CDCl₃, δ): 6.92 (bd, 1H), 4.57-4.62 (m, 1H), 3.98-4.12 (m,4H), 3.24-3.40 (m, 2H), 2.87-2.93 (m, 1H), 2.65 (s, 2H), 1.92-2.00 (m,1H), 1.66-1.75 (m, 2H), 1.32-1.40 (m, 2H), 1.27 (s, 12H), 0.89 (t, J=7Hz, 3H).

Preparation of Compound 14

[0143] To a stirred solution of the ketal 12 (487 mg; 1.36 mmol) in 12.0mL of methanol at room temperature was added 0.1N HCl solution (7.0 mL).The resulting cloudy mixture was warmed to reflux for 30 min. and thenallowed to cool to room temperature. The majority of the methanol wasthen removed in vacuo and the remaining solution extracted several timeswith ethyl acetate. The combined extracts were dried (MgSO₄), filteredand concentrated in vacuo. Flash chromatography on silica gel (eluent:85% ethyl acetate/hexane) provided 323 mg (76%) of the keto-amide 14 asa white solid, mp. 86-88° C.

[0144]¹H NMR (CDCl₃, δ): 7.51 (bd, 1H), 4.60 (m, 1H), 3.47 (s, 2H),3.24-3.44 (m, 2H), 2.82-2.90 (m, 1H), 2.54 (t, J=7 Hz, 2H), 1.98-2.09(m, 1H), 1.57-1.61 (m, 2H), 1.27 (s, 12H), 0.89 (t, J=7 Hz, 3H). Anal.calcd for C₁₆H₂₇NO₃S: C, 61.29; H, 8.70. Found: C, 61.21; H, 8.75. [α]²⁵_(D)−19⁰ (c=2, EtOH).

Preparation of Compound 15

[0145] To a stirred solution of PAI 13 (70 mg; 0.24 mmol) in 3.0 mL ofdry methanol under N₂ at −15° C. was added sodium borohydride (10 mg;0.26 mmol). After 1 h, another 10 mg of sodium borohydride was added andstirring allowed to continue for another hour. At this point, themixture was quenched with 1 mL of acetone and allowed to warm to roomtemperature. The solvent was removed in vacuo, the residue redissolvedin CH₂Cl₂ and washed with water. The organic layer was then dried(MgSO₄), filtered and concentrated in vacuo. The crude solid wasrecrystallized from ethyl acetate / hexane to give 39 mg. (55%) of thehydroxy-amide 15 as a mixture of 2 diastereomers.

[0146]¹H NMR (CDCl₃, δ): 6.48-6.60 (bm, 1H), 4.46-4.64 (M, 2H),4.27-4.38 (m, 1H), 4.00-4.06 (m, 1H), 2.81-2.90 (m, 1H), 2.08-2.52 (m,5H), 1.39-1.58 (m, 2H), 1.28 (s, 512H).

Preparation of Compound 17

[0147] To a stirred solution of the oxazolidinone 16 (Fluka, 388 mg; 3.0mmol) in 10 mL of dry THF under N₂ at −78° C. was added n-BuLi solution(1.38 mL of 2.4 mmol/mL solution) dropwise. At the end of the addition,the mixture was warmed to 0° C. for 10 min., quenched with decanoylchloride (0.81 mL; 3.9 mmol) and stirred for an additional 30 min. atthis temperature. At this point, the mixture was quenched with 2.0 mL ofsaturated NaHCO₃ solution and the solvent removed in vacuo. The residuewas taken up into CH₂Cl₂ and washed with 20% K₂CO₃ solution. The organiclayer was then dried (MgSO₄), filtered and the solvent removed in vacuo.Flash chromatography on silica gel (eluent: 35% ethyl acetate / hexane)provided 740 mg. (870%o) of oily oxazolidinone 17.

[0148]¹H NMR (CDCl₃, δ): 4.43-4.48 (m, 1H), 4.20-4.31 (m, 2H), 2.83-3.03(m, 2H), 2.36-2.42 (m, 1H), 1.62-1.69 (m, 2H), 1.28-1.33 (m, 12H),0.88-0.94 (m, 9H), Anal. calcd for C₁₆H₂₉O₃N: C, 67.79; H, 10.33. Found:C, 67.87; H, 10.59. [α]²⁵ _(D+)69⁰ (c=2, EtOH).

Preparation of Compound 21

[0149] To a stirred solution of trans-4-decanal (2.00 g; 13.0 mmol) andsulfamic acid (3.78 g; 38.9 mmol) in 80 mL of 3:1 THF:H₂O at 0° C. wasadded NaClO₂ solution (1.41 g; 15.6 mmol dissolved in 10 mL of water)dropwise. At the end of the addition, the mixture was allowed to warm toroom temperature for 10 min. At this point, most of the THF was thenremoved in vacuo. The resulting mixture was extracted several times withCH₂Cl₂. The combined extracts were dried (MgSO₄), filtered andconcentrated in vacuo. The residue was diluted with 10 mL of hexane,filtered and finally concentrated in vacuo to give 2.342 g of crude oilyacid 18. To a stirred solution of the crude acid (2.342 g) in 50 mL ofdry CH₂Cl₂ under N₂ at room temperature was added 2 drops of DMFfollowed by oxalyl chloride (1.56 mL; 17.9 mmol) dropwise. After 30min., gas evolution had ceased and the solvent was removed in vacuo toprovide the crude acid chloride 19. Crude 19 was then redissolved in 30mL of CH₂Cl₂ and Meldrums acid (1.99 g; 13.8 mmol) was added. Themixture was cooled to 0° C. and pyridine (2.79 mL; 34.5 mmol) wasdropwise. After the addition was complete, the mixture was allowed towarn to room temperature for 1.5 h then diluted with CH₂Cl₂ and washedwith 10% KHSO₄ solution. The organic layer was then dried (MgSO₄),filtered and concentrated in vacuo. The residue was then dissolved in 30mL of methanol and heated to reflux for 1 h, cooled and the solventremoved in vacuo. The oily residue was chromatographed on silica gel(eluent: 8% ethyl acetate / hexane) to give 558 mg (18%) of thebeta-ketoester 21 as a clear oil.

[0150] Keto tautomer: ¹H NMR (CDCl₃, δ): 5.37-4.58 (m, 2H), 3.76 (s,3H), 3.47 (s, 2H), 2.62 (t, J=7 Hz, 2H), 2.27-2.35 (m, 3H), 1.94-2.01(m, 2H), 1.23-1.38 (m, 6H), 0.90 (s, 3H).

Preparation of Compound 22

[0151] To a stirred solution of the beta-ketoester in benzene at roomtemperature was added ethylene glycol (0.64 mL; 11.4 mmol) followed by atrace of p-TSA. The mixture was then armed to reflux Dean-Stark). After4 h, another 0.64 mL of ethylene glycol was added. After a total of 23h, the mixture was allowed to cool to room temperature and washed with10% Na₂CO₃ solution. The organic layer was then dried (MgSO₄), filteredand concentrated in vacuo. Flash chromatography on silica gel (eluent:8% ethyl acetate/hexane provided 513 mg (50%) of the ketal intermediate22 together with 152 mg (18%) of recovered beta-ketoester 21.

[0152]¹H NMR (CDCl₃, δ): 5.39-5.49 (m, 2H), 3.99-4.01 (4H), 3.71 (s,3H), 2.69 (s, 2H), 1.86-2.15 (m, 7H), 1.29-1.37 (m, 6H), 0.90 (t, J=7Hz, 3H).

Preparation of Compound 24

[0153] To a stirred solution of the ketal (450 mg; 1.66 mmol) in 4.0 mLof methanol at room temperature was added LiOH solution (1.66 mL of 1.0M stock solution). The mixture was then warmed to reflux for 15 min. andthen allowed to cool. The solvent was removed in vacuo and the crudecarboxylate redissolved in 20 mL of water. To this solution was addedL-homoserine lactone hydrochloride (251 mg; 1.83 mmol) followed by1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (350 mg;1.83 mmol) and the mixture allowed to stir for 6 h at room temperature.At this point, the mixture was extracted several times with ethylacetate. The combined extracts were dried (MgSO₄), filtered andconcentrated in vacuo to give 531 mg of crude amide 24.

[0154]¹H NMR (CDCl₃, δ): 7.00 (d, J=6 Hz, 1H), 5.38-5.44 (m,2H),4.55-4.65 (m, 1H), 4.45-4.51 (m, 1H), 4.28-4.31 (m, 1H), 3.99-4.12(m, 4H), 2.79-2.83 (m, 1H), 2.67 (s, 2H), 1.74-2.19 (m, 7H), 1.29-1.36(m, 6H), 0.89 (t, J=7 Hz, 3H).

Preparation of Compound 25

[0155] The crude amide 24 was dissolved in 4.0 mL of methanol at roomtemperature and 3.0 mL of 0.1 N HCl solution was added. The resultingmixture was warmed to reflux for 1 h, cooled to room temperature and themajority of the methanol removed in vacuo. The remaining aqueous phasewas extracted several times with ethyl acetate. The combined extractswere dried (MgSO₄), filtered and concentrated in vacuo. Flashchromatography on silica gel (eluent: 75% ethyl acetate / hexane) gave175 mg (36%) of the unsaturated keto-amide 25, mp. 92-93° C.

[0156]¹H NMR (CDCl₃, δ): 7.70 (d, J=4.3 Hz, 1H), 5.32-5.50 (m, 2H),4.56-4.65 (m, 1H), 4.46-4.52 (m, 1H), 4.25-4.33 (m, 1H), 3.48 (s, 2H),2.72-2.81 (m, 1H), 2.58-2.63 (m, 2H), 2.17-2.32 (m, 3H), 1.93-1.98 (m,2H), 1.25-1.38 (m, 6H), 0.89 (t, J=7 Hz, 3H). [α]²⁵ _(D)−23⁰ (c=2,EtOH).

Preparation of Compound 27

[0157] Prepared by coupling of the aminolactam 26 (129 mg; 0.94 mmol)and the lithium salt derived from the ester 10 (278 mg; 1.02 mmol) inwater (2.0 mL) in the presence of carbodiimide 4 (180 mg; 0.94 mmol)according to the procedure used for the preparation of compound 11. Thecrude product (293 mg) was then dissolved in 7.0 mL of methanol and 3.0mL of 0.1 N HCl was added and the mixture heated to reflux for 1 h andthen allowed to cool to room temperature and then processed as forcompound 14. The crude solid product was recrystallized from ethylacetate/hexane to give 150 mg (54%) of amide 27, mp. 158-160° C.

[0158]¹H NMR (CDCl₃, δ): 7.60 (bm, 1H), 6.45 (bm, 1H), 4.45 (M, 1H),3.47 (s, 2H), 3.38-3.42 (m, 2H), 2.69-2.77 (m, 1H), 2.55 (t, J=7 Hz,2H), 1.96-2.04 (m, 1H), 1.58-1.61 (m, 2H), 1.27 (s, 12H), 0.89 (t, J=7Hz, 3H).

Preparation of Compound 29

[0159] A solution of maleic anhydride (12.2 mg, 0.125 mmol) indichloromethane (0.2 mL) was added to a solution of DL homoserinelactone (12.6 mg, 0.125 mmol) in dichloromethane (0.1 mL). As themixture was stirred, a white precipitate formed rapidly, became oilyover 10 min, then became a granular solid over the next 10 min..Stirring was continued for a total of 75 min, and the mixture was thendiluted with di-isopropyl ether (2 mL) and the solid was removed byfiltration to give N-maleoylhomoserine lactone (20 mg, 81%).

[0160]¹H NMR (DMSO) 13.3 (br s, 1 H), 9.12 (br d, J=8.8 Hz, 1 H), 6.30,6.24 (2 d, J=12.2 Hz, 1 H each), 4.64 (ddd, J=10.5, 9.1, 7.9 Hz, 1 H),4.36 (ddd, J=8.8, 8.8, 1.9 Hz, 1 H, 4.23 (ddd, I=10.5, 8.7, 6.5 Hz, 1H), 2.43 (dddd, J=12.2, 9.1, 6.4, 1.9 Hz, 1 H), 2.19 (dddd, J=12.2, 8.8,8.7, 7.9 Hz, 1 H). 13C NMR (DMSO) 174.8(s), 166.5(s), 164.8(s),131.7(d), 130.2(d), 65.5(t), 48.2(d), 21.8(t).

7. EXAMPLE Production of BAI Antibodies

[0161] Production of anti-autoinducer antibodies and experimental invivo therapeutic evaluation of said anti-autoinducer antibodies isdescribed in the sections that follow. Specifically, antibodies weremade to immunogenic conjugates containing PAI-1 covalently bound tobovine serum albumin, however, one of ordinary skill in the art willeasily recognize that other autoinducer molecules and additionalconjugates may be used in a similar procedure to produce variouscombinations of immunogenic conjugates and antibodies that recognizesaid immunogenic conjugates. One of ordinary skill in the art will alsorecognize that additional animal models can be used to assay thetherapeutic and diagnostic effectiveness of the anti-autoinducerantibodies.

[0162] 7.1 PAI's Role in Virulence

[0163] The role of PAI-1 and PAI-2 in colonization of Pseudomonasaeruginosa in neonatal mice was examined. BALB/cBy, mice 7-10 days old,were inoculated intranasally with approximately 10⁸ CFU of either wildtype or deletion mutants of P. aeruginosa. The lasI gene product is asynthase required for PAI-1 production and the rhII gene product isrequired for production of PAI-2 (Passador et al., 1996, J. Bact.178:5995-6000). The deletion strains were: PAO1, wild type; PAO-JP2,bearing a double deletion of lasI/rhII; PAO-JP1, bearing a lasIdeletion; and PAO-JP2/pJPP42, bearing a double deletion lasI/rhII butcarrying a complementing plasmid expressing both lasI and rhII.Bacterial suspensions of these strains were given in 2 μl aliquotsdirectly into the mouse nares until a total of 10 μl had beenadministered. Twenty-four hours after challenge, mice were sacrificed,and the right lung cultured for bacterial load and the left lung fixedin 10% buffered formalin for histological analysis. Pneumonia wasdefined as ≧100 CFU of Pseudomonas aeruginosa in the lung. Bacteremiawas defined as any bacteria cultured from the spleen.

[0164] The results indicate that mice inoculated with strain PAO-JP1,which does not produce PAI-1, exhibited a significant decrease in therates of pneumonia, bacteremia, and mortality in comparison to PAO1(FIG. 1). Moreover, mice inoculated with strain PAO-JP2, which producesneither PAI-1 nor PAI-2, exhibited an additional decrease in the ratesof pneumonia, bacteremia, and mortality over the already reducedinfection rate of PAO1. In sharp contrast, mice inoculated withPseudomonas aeruginosa JP2/pJPP42, which produces PAI-1 and PAI-2 byvirtue of a genetically complementing plasmid, exhibited pneumonia,bacteremia, and mortality rates comparable to wild type, PAO1 (FIG. 1).

[0165] 7.2 Anti-PAI Polyclonal Antibodies

[0166] The Pseudomonas aeruginosa autoinducer PAI-1 (structure on page16) and Compound D (structure on page 9) were separately conjugated tobovine serum albumin (BSA). These conjugates were used to immunizedifferent BALB/c mice. Initial injections of 50 μg of conjugate wereadministered subcutaneously in Freund's complete adjuvant and two weekslater were boosted with a second subcutaneous injection in Freund'sincomplete adjuvant. After two additional weeks, mice were tail bled andserum was isolated. A sandwich enzyme-linked immunosorbent assay (ELISA)utilizing a PAI-1 ovalbumin (OVA) conjugate was used to test for theproduction of specific serum antibodies that recognize autoinducers.Serum samples with an optical density (OD) that was five to ten foldhigher than serum from mice immunized with BSA only were consideredpositive.

[0167] 7.3 Neutralization of PAI with Antibodies

[0168] In Vitro neutralization of PAI-1 with anti-PAI-1 polyclonalantibodies. Immune serum, collected in Section 7.2, were tested forneutralization of PAI-1 in an in vitro ,bioassay. The E. coli MG4strain, containing the lysogen λI₁4 (a lasI/lasZ transcriptional fusion)and pPCS1 (a plasmid expressing lasR), was used as a positive control todetect the presence of PAI-1. Normally, when PAI-1 is added to culturesit can bind LasR, the PAI-1 specific transcriptional activator protein,and form a complex that is able to induce transcription of the lasI/lasZfusion protein. The production of β-galactosidase in this system is aquantitative and direct measure of the activation induced by PAI-1. Theexpression construct, λI₁4-MG4 (PPCS1), has been shown to havehalf-maximal expression at PAI-1 concentrations of 100 pM and can beactivated at PAI-1 concentrations as low as 10 pM (Seed et al., 1995, J.Bacteriol. 177:654-59, which is hereby incorporated by reference).

[0169] A test sample containing 100 pM of PAI-1 was preincubated for onehour at 37° C. with a 1:10 dilution of serum from mice immunized with aPAI-1 conjugate or a Compound D conjugate (both contain anti-PAI-1polyclonal antibodies). Control samples containing 100 pM PAI-1 wereincubated at 37° C. for one hour with a 1:10 dilution of preimmune serumor an equal volume of PBS. Following preincubation, the samples weretested in an E. coli bioassay using λ₁4-MG4 (pPCS1). When the bacteriain each sample reached an OD600 of 0.8-1.0 the samples were assayed forthe production of β-galactosidase which was expressed as Miller Units ofactivity. The test samples preincubated with serum from immune micedisplayed a 70% reduction in β-galactosidase production as compared tocontrol sample preincubated with nonimmune serum (FIG. 2). These resultsindicate that PAI-1 conjugates or related conjugates (Compound D) caninduce the production of polyclonal antibodies that can react with PAI-1and inhibit its interaction with LasR.

[0170] Mice immunized with the PAI-1 conjugate were used to producemonoclonal antibodies. These antibodies were screened using an ELISAutilizing a PAM-1/OVA conjugate. Positive clones were tested in aPseudomonas aeruginosa bioassay. PAO-JP2 (bearing the lasI/rhII doubledeletion) produces no PAI-1 but retains the ability to produce LasR.When PAI-1 is added exogenously, it can bind to LasR and induce thetranscription of lasI. Test samples containing 40 nM PAI (theconcentration that stimulates half-maximal activity in this assay) werepreincubated at 37° C. with anti-PAI-1 monoclonal antibody (618.4).Control samples containing 40 nm PAI-1 were preincubated at 37° C. withan equal volume of PBS. Following preincubation, the samples were testedin a Pseudomonas aeruginosa bioassay using PAO-JP2 containing a plasmidwith a lasI/LacZ fusion. When the bacteria reached on OD₆₀₀ of 0.8-1.0,the samples were assayed for the production of β-galactosidase, whichwas expressed as Miller Units of activity. The test sample preincubatedwith anti-PAI-1 monoclonal antibody 618.4 displayed an 80% reduction inthe production of β-galactosidase as compared to the control sample(FIG. 3). These results indicate that in Pseudomonas aeruginosaantibodies specific for PAI-1 can inhibit PAI-1 activation of LasR andtranscription of other genes that are regulated by LasR/PAI-1.

[0171] Although the invention has been described in detail for thepurposes of illustration, it is understood that such detail is solelyfor that purpose, and variations can be made therein by those skilled inthe art without departing from the spirit and scope of the inventionwhich is defined by the following claims.

What is claimed is:
 1. An immunogenic conjugate comprising a carriermolecule covalently conjugated or otherwise bound to an autoinducer of aGram negative bacteria of a compound of Formula (I):

where X is O, S, N—(C₁-C₆) alkyl, NR², N-phenyl; Y is C₁-C₆ straight orbranched alkyl, C₁-₆ straight or branched alkenyl, C₁-C₆ straight orbranched alkynyl; Z is C═O, C═S, CHOH, C═N—NR¹, C═N—OH, C₁-₈ straight orbranched alkyl, C₁-₈ straight or branched alkenyl, C₁-C₈ straight orbranched alkynyl; L is C₁-C₁₈ straight or branched alkyl, C₁-C₁₈straight or branched alkenyl, C₁-C₁₈ straight branched alkynyl, or—CO₂H, —CO₂R¹, —CHO, —C≡N, —N═C═O, —N═C═S, OH, OR¹, —CH═CH—CH₂Br,—CH═CH—CH₂Cl, —SAc or SH, where R¹ is C₁-C₆ straight or branched alkyl,m is 0 or 1; z is 0 or 1; R² is H, C₁-C₆ straight or branched alkylC₁-C₆ straight or branched alkenyl or C₁-C₆ straight or branchedalkynyl, or CO₂H; and Q is CH or N; and n is 0-3 with the proviso thatwhen n is 0, X is N-C₁-₆ alkyl) or N-phenyl.
 2. An immunogenic conjugateaccording to claim 1, wherein said carrier molecule comprises alysine-containing protein.
 3. An immunogenic conjugate according toclaim 2, wherein said lysine-containing protein is selected from thegroup consisting of bovine serum albumin, chicken egg ovalbumin, keyholelimpet hemocyanin, tetanus toxoid, diphtheria toxoid, and thyroglobulin.4. An immunogenic conjugate according to claim 1, wherein saidautoinducer is produced by a Gram negative bacteria comprising Aeromonashydrophila, Agrobacterium tumefaciens, Burkholderia cepacia,Chromobacterium violaceum, Enterobacter agglomerans, Erwinia stewarti,Erwinia carotovora, Escherichia coli, Nitrosomas europea, Photobacteriumfischeri, Pseudomonas aeruginosa, Pseudomonas aureofaciens, Rhizobiumleguminosarum, Serratia liquefaciens, or Vibrio harveyi.
 5. Animmunogenic conjugate according to claim 1, wherein said autoinducercomprises N-(3-oxododecanoyl)-L-homoserine lactone,N-(butanoyl)-L-homoserine lactone, N-hexanoyl-homoserine lactone,N-(3-oxohexanoyl)-homoserine lactone, N-β(hydroxybutyryl)-homoserinelactone, N-(3-oxooctanoyly-L-homoserine lactone, orN-(3R-hydroxy-cis-tetradecanoyl)-L-homoserine lactone.
 6. Theimmunogenic conjugate of claim 6 in which the autoinducer isN-(3-oxododecanoyl)-L-homoserine lactone or N-(butanoyl)-L-homoserinelactone.
 7. An immunogenic conjugate of claim 1, wherein said carriermolecule has at least one amine group, said autoinducer has an N-acylhomoserine lactone structure, and said conjugate is the reductiveamination product of said carrier molecule and said autoinducer.
 8. Anisolated antibody or fragment thereof which specifically binds anautoinducer of or produced by a Gram negative bacteria.
 9. The isolatedantibody or fragment thereof of claim 8 in which the autoinducer is acompound of Formula (I):

where X is O, S, N—(C₁-C₆) alkyl, NR², N-phenyl; Y is C₁-C₆ straight orbranched alkyl, C₁-C₆ straight or branched alkenyl, C₁-C₆ straight orbranched alkynyl; Z is C═O, C═S, CHOH, C═N—NR¹, C═N—OH, C₁-C₈ straightor branched alkyl, C₁-C₈ straight or branched alkenyl, C₁-C₈ straight orbranched alkynyl; L is C₁-C₁₈ straight or branched alkyl, C₁-C₈ straightor branched alkenyl, C₁-C₁₈ straight branched alkynyl, or —CO₂H, —CO₁R¹,—CHO, —C≡N, —N═C═O, —N═C═S, OH, OR¹, —CH═CH—CH₂Br, —CH═CH—CH₂Cl, —SAc orSH, where R¹ is C₁-C₆ straight or branched alkyl, m is 0 or 1; z is 0 or1; R² is H, C₁-C₆ straight or branched alkyl, C₁-C₆ straight or branchedalkenyl or C₁-C₆ straight or branched alkynyl, or CO₂H; and Q is CH orN; and n is 0-3 with the proviso that when n is 0, X is N—(C₁-C₆ alkyl)or N-phenyl.
 10. The isolated antibody or fragment thereof of claim 8which is a monoclonal antibody.
 11. The isolated antibody or fragmentthereof of claim 8 in which the autoinducer comprisesN-(3-oxododecanoyl)-L-homoserine lactone, N-(butanoyl)-L-homoserinelactone, N-hexanoyl-homoserine lactone, N-(3-oxohexanoyl)-homoserinelactone, N-β(hydroxybutyryl)-homoserine lactone,N-(3-oxooctanoyl)-L-homoserine lactone, orN-(3R-hydroxy-cis-tetradecanoyl)-L-homoserine lactone.
 12. The isolatedantibody or fragment thereof of claim 11 in which the autoinducer isN-(3-oxododecanoyl)-L-homoserine lactone or N-(butanoyl)-L-homoserinelactone.
 13. The isolated antibody or fragment thereof of claim 8 inwhich the autoinducer is covalently conjugated or otherwise bound to acarrier molecule.
 14. The isolated antibody or fragment thereof of claim13 in which the carrier molecule is selected from the group consistingof bovine serum albumin, chicken egg ovalbumin, keyhole limpethemocyanin, tetanus toxoid, diphtheria toxoid, and thyroglobulin. 15.The isolated antibody or fragment thereof of claim 8 in which theautoinducer is produced by a Gram negative bacteria comprising Aeromonashydrophila, Agrobacterium tumefaciens, Burkholderia cepacia,Chromobacterium violaceum, Enterobacter agglomerans, Erwinia stewarti,Erwinia carotovora, Escherichia coli, Nitrosomas europea, Photobacteriumfischeri, Pseudomonas aeruginosa, Pseudomonas aureofaciens, Rhizobiumleguminosarum, Serratia liquefaciens, or Vibrio harveyi.
 16. A methodfor detecting a Gram negative bacteria autoinducer in a samplecomprising adding to the sample an antibody in which the antibodyspecifically binds the autoinducer of a Gram negative bacteria of acompound of Formula (I):

where X is O, S, N—(C₁-C₆) alkyl, NR², N-phenyl; Y is C₁-C₆ straight orbranched alkyl, C₁-C₆ straight or branched alkenyl, C₁-C₆ straight orbranched alkynyl; Z is C═O, C═S, CHOH, C═N—NR¹, C═N—OH, C₁-C₈ straightor branched alkyl, C₁-C₈ straight or branched alkenyl, C₁-C₈ straight orbranched alkynyl; L is C₁-C₈ straight or branched alkyl, C₁-C₁₈ straightor branched alkenyl, C₁-C₁₈ straight branched allynyl, or —CO₂H, —CO₂R¹,—CHO, —C≡N, —N═C═O, —N═C═S, OH, OR¹, —CH═CH—CH₂Br, —CH═CH—CH₂Cl, —SAc orSH, where R¹ is C₁-C₆ straight or branched alkyl, m is 0 or 1; z is 0 or1; R² is H, C₁-C₆ straight or branched alkyl, C₁-C₆ straight or branchedalkenyl or C₁-C6 straight or branched alkynyl, or CO₂H; and Q is CH orN; and n is 0-3 with the proviso that when n is 0, X is N—(C₁-C₆ alkyl)or N-phenyl.
 17. The method according to claim 16 wherein theautoinducer is produced by a Gram negative bacteria comprising Aeromonashydrophila, Agrobacterium tuinetaciens, Burkholderia cepacia,Chromobacterium violaceum, Enterobacter agglomerans, Erwinia stewarti,Erwinia carotovora, Escherichia coli, Nitrosomas europea, Photobacteriumfischeri, Pseudomonas aeruginosa, Pseudomonas aureofaciens, Rhizobiumleguminosarum, Serratia liquefaciens, or Vibrio harveyi.
 18. A method oftreating or preventing an infectious disease in a subject comprisingadministering an amount of an immunogenic conjugate in which theimmunogenic conjugate comprises a carrier molecule covalently conjugatedor otherwise bound to an autoinducer of a Gram negative bacteria of acompound of Formula (I):

where X is O, S, N—(C₁-₆) alkyl, NR², N-phenyl; Y is C₁-C₆ straight orbranched alkyl, C₁-C₆ straight or branched alkenyl, C₁-C₆ straight orbranched alkynyl; Z is C═O, C═S, CHOH, C═N—NR¹, C═N—OH, C₁-C₈ straightor branched alkyl, C₁-C₈ straight or branched alkenyl, C₁-C₈ straight orbranched alkynyl; L is C₁-C₁₈ straight or branched alkyl, C₁-C₁₈straight or branched alkenyl, C₁-C₁₈ straight branched alkynyl, or—CO₂H, —CO₂R¹, —CHO, —C≡N, —N═C═O, —N═C═S, OH, OR¹, —CH═CH—CH₂Br,—CH═CH—CH₂Cl, —SAc or SH, where R¹ is C₁-C₆ straight or branched alkyl,m is 0 or 1; z is 0 or 1; R² is H, C₁-C₆ straight or branched alkyl,C₁-C₆ straight or branched alkenyl or C₁-C₆ straight or branchedalkynyl, or CO₂H; and Q is CH or N; and n is 0-3 with the proviso thatwhen n is 0, X is N—(C₁C₆ alkyl) or N-phenyl, in which said amount iseffective to treat or prevent said infectious disease.
 19. The methodaccording to claim 18 wherein said immunogenic conjugate is administeredorally, intradermally, intramuscularly, intraperitoneally,intravenously, subcutaneous, or intranasally.
 20. The method accordingto claim 18 wherein said subject is a human.
 21. The method of claim 18in which the infectious disease is caused by a Gram negative bacteria.22. A method of treating or preventing an infectious disease in asubject comprising administering an amount of an antibody or fragmentthereof which specifically binds an autoinducer of a Gram negativebacteria of a compound of Formula (I):

where X is O, S, N—(C₁-C₆) alkyl, NR², N-phenyl; Y is C₁-C₆ straight orbranched alkyl, C₁-C₆ straight or branched alkenyl, C₁-C₆ straight orbranched alkynyl; Z is C═O, C═S, CHOH, C═N—NR¹, C═N—OH, C₁-C₈ straightor branched alkyl, C₁-C₈ straight or branched alkenyl, C₁-C₈ straight orbranched alkynyl; L is C₁-C₁₈ straight or branched alkyl, C₁-C₁₈straight or branched alkenyl, C₁-C₁₈ straight branched alkynyl, or—CO₂H, —CO₂R¹, —CHO, —C≡N, —N═C═O, —N═C═S, OH, OR¹, —CH═CH—CH₂Br,—CH═CH—CH₂Cl, —SAc or SH, where R¹ is C₁-C₆ straight or branched alkyl,m is 0 or 1; z is 0 or 1; R² is H, C₁-C₆ straight or branched alkyl,C₁-C₆ straight or branched alkenyl or C₁-C₆ straight or branchedalkynyl, or CO₂H; and Q is CH or N; and n is 0-3 with the proviso thatwhen n is 0, X is N-(C₁-C₆ alkyl) or N-phenyl, in which said amount iseffective to treat or prevent said infectious disease.
 23. The methodaccording to claim 22 wherein said subject is a human.
 24. The methodaccording to claim 22 wherein said antibody is administered orally,intradermally, intramuscularly, intraperitoneally, intravenously,subcutaneously, or intranasally.
 25. The method of claim 22 in which theinfectious disease is caused by a Gram negative bacteria.
 26. Adiagnostic kit comprising an antibody which specifically binds anautoinducer of a Gram negative bacteria of a compound of Formula (I):

where X is O, S, N—(C₁-C₆) alkyl, NR², N-phenyl; Y is C₁-C₆ straight orbranched alkyl, C₁-C₆ straight or branched alkenyl, C₁-C₆ straight orbranched alkynyl; Z is C═O, C═S, CHOH, C═N—NR¹, C═N—OH, C₁-C₈ straightor branched alkyl, C₁-C₈ straight or branched alkenyl, C₁-C₈ straight orbranched alkynyl; L is C₁-C₁₈ straight or branched alkyl, C₁-C₁₈straight or branched alkenyl, C₁-C₁₈ straight branched alkynyl, or—CO₂H, —CO₂R¹, —CHO, —C≡N, —N═C═O, —N═C═S, OH, OR¹, —CH═CH—CH₂Br,—CH═CH—CH₂Cl, —SAc or SH, where R¹ is C₁-C₆ straight or branched alkyl,m is 0 or 1; z is 0 or 1; R² is H, C₁-C₆ straight or branched alkyl,C₁-C₆ straight or branched alkenyl or C₁-C₆ straight or branchedalkynyl, or CO₂H; and Q is CH or N; and n is 0-3 with the proviso thatwhen n is 0, X is N-(C₁-C₆ alkyl) or N-phenyl.
 27. A pharmaceuticalcomposition comprising an antibody or fragment thereof whichspecifically binds an autoinducer produced by a Gram negative bacteria;and a pharmaceutically acceptable carrier.
 28. The pharmaceuticalcomposition of claim 27 in which the autoinducer is a compound ofFormula (I):

where X is O, S, N—(C₁-C₆) alkyl, NR², N-phenyl; Y is C₁-C₆ straight orbranched alkyl, C₁-C₆ straight or branched alkenyl, C₁-C₆ straight orbranched alkynyl; Z is C═O, C═S, CHOH, C═N—NR¹, C═N—OH, C₁-C₈ straightor branched alkyl, C₁-C₈ straight or branched alkenyl, C₁-C₈ straight orbranched alkynyl; L is C₁-C₁₈ straight or branched alkyl, C₁-C₁₈straight or branched alkenyl, C₁-C₁₈ straight branched alkynyl, or—CO₂H, —CO₂R¹, —CHO, —C≡N, —N═C═O, —N═C═S, OH, OR¹, —CH═CH—CH₂Br,—CH═CH—CH₂Cl, —SAc or SH, where R¹ is C₁-C₆ straight or branched alkyl,m is 0 or 1; z is 0 or 1; R² is H, C₁-C₆ straight or branched alkyl,C₁-C₆ straight or branched alkenyl or C₁-C₆ straight or branchedalkynyl, or CO₂H; and Q is CH or N; and n is 0-3 with the proviso thatwhen n is 0, X is N-(C₁-C₆ alkyl) or N-phenyl; and a pharmaceuticallyacceptable carrier.
 29. The pharmaceutical composition of claim 27 whichthe antibody is a monoclonal antibody.
 30. The pharmaceuticalcomposition of claim 27 in which the autoinducer comprisesN-(3-oxododecanoyl)-L-homoserine lactone, N-(butanoyl)-L-homoserinelactone, N-hexanoyl-homoserine lactone, N-(3-oxohexanoyl)-homoserinelactone, N-β(hydroxybutyryl)-homoserine lactone,N-(3-oxooctanoyl)-L-homoserine lactone, orN-(3R-hydroxy-cis-tetradecanoyl)-L-homoserine lactone.
 31. Thepharmaceutical composition of claim 27 in which the autoinducer isN-(3-oxododecanoyl)-L-homoserine lactone or N-(butanoyl)-L-homoserinelactone.
 32. The pharmaceutical composition of claim 27 in which theautoinducer is covalently conjugated or otherwise bound to a carriermolecule.
 33. The pharmaceutical composition of claim 32 in which thecarrier molecule is selected from the group consisting of bovine serumalbumin, chicken egg ovalbumin, keyhole limpet hemocyanin, tetanustoxoid, diphtheria toxoid, and thyroglobulin.
 34. The pharmaceuticalcomposition of claim 27 in which the autoinducer is produced by a Gramnegative bacteria comprising Aeromonas hydrophila, Agrobacteriumtumefaciens, Burkholderia cepacia, Chromobacterium violaceum,Enterobacter agglomerans, Erwinia stewarti, Erwinia carotovora,Escherichia coli, Nitrosomas europea, Photobacterium fischeri,Pseudomonas aeruginosa, Pseudomonas aureofaciens, Rhizobiumleguminosarum, Serratia liquefaciens, or Vibrio harveyi.